| anno_start anno_end anno_text entity_type sentence section |
| 40 64 autocatalytic activation ptm A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome TITLE |
| 72 86 20S proteasome complex_assembly A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome TITLE |
| 18 32 20S proteasome complex_assembly Biogenesis of the 20S proteasome is tightly regulated. ABSTRACT |
| 15 26 propeptides structure_element The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT |
| 42 53 active-site site The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT |
| 54 64 threonines residue_name The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT |
| 69 86 autocatalytically ptm The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT |
| 68 87 strict conservation protein_state However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. ABSTRACT |
| 91 100 threonine residue_name However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. ABSTRACT |
| 12 23 mutagenesis experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 25 46 X-ray crystallography experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 51 69 biochemical assays experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 86 91 Lys33 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 129 139 propeptide structure_element Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 161 165 Thr1 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 218 223 Asp17 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 238 253 catalytic triad site Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT |
| 0 12 Substitution experimental_method Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 16 20 Thr1 residue_name_number Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 24 27 Cys residue_name Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 58 63 Lys33 residue_name_number Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 68 79 inactivates protein_state Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 84 94 proteasome complex_assembly Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT |
| 11 18 Thr1Ser mutant Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT |
| 19 25 mutant protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT |
| 29 35 active protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT |
| 72 81 wild type protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT |
| 125 129 Ser1 residue_name_number Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT |
| 72 92 propeptide autolysis ptm This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT |
| 147 157 proteasome complex_assembly This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT |
| 170 188 threonine protease protein_type This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT |
| 5 15 proteasome complex_assembly The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. ABSTRACT |
| 54 72 threonine protease protein_type The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. ABSTRACT |
| 85 95 proteasome complex_assembly Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. ABSTRACT |
| 96 107 active site site Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. ABSTRACT |
| 4 32 20S proteasome core particle complex_assembly The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO |
| 34 36 CP complex_assembly The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO |
| 49 71 non-lysosomal protease protein_type The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO |
| 75 85 eukaryotic taxonomy_domain The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO |
| 20 21 α protein Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO |
| 42 52 β subunits protein Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO |
| 72 82 heptameric oligomeric_state Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO |
| 83 88 rings structure_element Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO |
| 130 145 hollow cylinder structure_element Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO |
| 10 18 inactive protein_state While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO |
| 19 29 α subunits protein While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO |
| 50 55 rings structure_element While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO |
| 61 71 β subunits protein While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO |
| 87 92 rings structure_element While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO |
| 38 48 β subunits protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 57 59 β1 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 61 63 β2 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 68 70 β5 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 88 114 proteolytic active centres site Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 127 129 CP complex_assembly Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 164 175 propeptides structure_element Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO |
| 21 23 CP complex_assembly In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 40 51 prosegments structure_element In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 56 81 autocatalytically removed ptm In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 117 136 active site residue site In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 137 141 Thr1 residue_name_number In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 182 189 Gly(-1) residue_name_number In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO |
| 15 26 propeptides structure_element Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO |
| 50 56 active protein_state Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO |
| 57 59 CP complex_assembly Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO |
| 36 61 substrate-binding channel site Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO |
| 129 135 active protein_state Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO |
| 136 146 β subunits protein Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO |
| 152 164 active sites site Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO |
| 23 27 Thr1 residue_name_number Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate. INTRO |
| 19 26 complex complex_assembly Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO |
| 61 66 water chemical Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO |
| 92 98 enzyme complex_assembly Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO |
| 123 130 peptide chemical Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO |
| 4 14 proteasome complex_assembly The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 40 80 N-terminal nucleophilic (Ntn) hydrolases protein_type The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 90 94 free protein_state The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 121 125 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 158 162 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 205 209 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO |
| 40 74 autocatalytic precursor processing ptm This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO |
| 90 98 immature protein_state This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO |
| 99 111 active sites site This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO |
| 113 117 Thr1 residue_name_number This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO |
| 152 159 Gly(-1) residue_name_number This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO |
| 47 51 Thr1 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO |
| 88 93 Lys33 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO |
| 110 126 hydrogen-bonding bond_interaction An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO |
| 139 143 Thr1 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO |
| 63 84 autocatalytic removal ptm In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. INTRO |
| 92 102 propeptide structure_element In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. INTRO |
| 69 79 proteasome complex_assembly Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. INTRO |
| 80 91 active-site site Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. INTRO |
| 10 45 biochemical and structural analyses experimental_method Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 91 93 β5 protein Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 94 104 propeptide structure_element Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 113 124 active site site Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 228 252 autocatalytic activation ptm Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 260 270 proteasome complex_assembly Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO |
| 44 47 Thr residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO |
| 53 56 Cys residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO |
| 60 63 Ser residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO |
| 101 122 X-ray crystallography experimental_method Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO |
| 137 167 activity and inhibition assays experimental_method Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO |
| 16 26 proteasome complex_assembly Inactivation of proteasome subunits by T1A mutations RESULTS |
| 27 35 subunits protein Inactivation of proteasome subunits by T1A mutations RESULTS |
| 39 42 T1A mutant Inactivation of proteasome subunits by T1A mutations RESULTS |
| 43 52 mutations experimental_method Inactivation of proteasome subunits by T1A mutations RESULTS |
| 0 10 Proteasome complex_assembly Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. RESULTS |
| 131 141 eukaryotic taxonomy_domain Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. RESULTS |
| 20 31 active site site Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 32 36 Thr1 residue_name_number Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 40 51 mutation to experimental_method Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 52 55 Ala residue_name Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 126 136 proteasome complex_assembly Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 137 149 active sites site Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS |
| 0 5 Yeast taxonomy_domain Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 44 50 β1-T1A mutant Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 54 60 β2-T1A mutant Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 128 137 catalytic protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 138 148 β subunits protein Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 153 161 disabled protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 166 183 carry remnants of protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 201 212 propeptides structure_element Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS |
| 32 34 β1 protein These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. RESULTS |
| 39 41 β2 protein These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. RESULTS |
| 17 20 T1A mutant By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. RESULTS |
| 41 43 β5 protein By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. RESULTS |
| 29 35 β5-T1A mutant Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 52 62 propeptide structure_element Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 64 66 pp chemical Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 68 100 deleted but expressed separately experimental_method Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 104 109 trans protein_state Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 111 117 β5-T1A mutant Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 118 120 pp chemical Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 121 126 trans protein_state Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS |
| 12 37 crystallographic analysis experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 45 51 β5-T1A mutant Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 52 54 pp chemical Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 55 60 trans protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 61 67 mutant protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 90 98 mutation experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 138 159 catalytic active site site Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 173 188 trans-expressed experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 189 191 β5 protein Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 192 202 propeptide structure_element Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 206 215 not bound protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 223 225 β5 protein Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 226 251 substrate-binding channel site Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS |
| 33 39 β5-T1A mutant The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS |
| 40 46 mutant protein_state The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS |
| 47 49 pp chemical The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS |
| 50 53 cis protein_state The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS |
| 0 26 Sequencing of the plasmids experimental_method Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 59 64 yeast taxonomy_domain Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 88 113 site-directed mutagenesis experimental_method Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 132 138 β5-T1A mutant Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 139 145 mutant protein_state Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 146 148 pp chemical Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 149 152 cis protein_state Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS |
| 48 52 K81R mutant We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 64 66 β5 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 112 143 This single amino-acid exchange experimental_method We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 162 171 interface site We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 188 190 α4 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 192 194 β4 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 199 201 β5 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 251 253 CP complex_assembly We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS |
| 34 45 β5-T1A-K81R mutant The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 46 52 mutant protein_state The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 77 94 crystal structure evidence The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 100 105 yeast taxonomy_domain The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 106 116 proteasome complex_assembly The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 118 121 yCP complex_assembly The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 132 138 β5-T1A mutant The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS |
| 0 10 Propeptide structure_element Propeptide conformation and triggering of autolysis RESULTS |
| 42 51 autolysis ptm Propeptide conformation and triggering of autolysis RESULTS |
| 22 32 proteasome complex_assembly In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS |
| 49 60 propeptides structure_element In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS |
| 65 90 autocatalytically cleaved ptm In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS |
| 100 106 mature protein_state In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS |
| 107 124 β-subunit domains protein In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS |
| 12 14 β1 protein For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 73 83 propeptide structure_element For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 104 108 (-2) residue_number For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 147 168 S1 specificity pocket site For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 176 201 substrate-binding channel site For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 223 225 45 residue_number For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS |
| 38 48 prosegment structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 58 78 antiparallel β-sheet structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 86 97 active site site Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 108 115 Gly(-1) residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 125 144 γ-turn conformation structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 188 201 hydrogen bond bond_interaction Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 210 217 Leu(-2) residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 223 227 Thr1 residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS |
| 27 33 β1-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 34 40 mutant protein_state Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 41 61 crystallographically experimental_method Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 93 103 structures evidence Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 111 117 β2-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 129 142 β1-T1A-β2-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS |
| 11 13 β1 protein In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS |
| 29 36 Gly(-1) residue_name_number In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS |
| 52 62 sharp turn structure_element In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS |
| 81 100 prosegment cleavage ptm In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS |
| 13 32 γ-turn conformation structure_element However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS |
| 52 65 hydrogen bond bond_interaction However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS |
| 198 205 Gly(-1) residue_name_number However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS |
| 233 237 Thr1 residue_name_number However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS |
| 14 16 β2 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 17 27 propeptide structure_element Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 29 36 Thr(-2) residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 50 59 S1 pocket site Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 102 109 Leu(-2) residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 113 115 β1 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 156 158 β2 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 159 168 S1 pocket site Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 180 185 Gly45 residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS |
| 0 7 Thr(-2) residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS |
| 18 25 Gly(-1) residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS |
| 31 47 hydrogen bonding bond_interaction Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS |
| 112 116 Thr1 residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS |
| 38 40 β5 protein Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS |
| 41 51 propeptide structure_element Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS |
| 59 70 β5-T1A-K81R mutant Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS |
| 71 77 mutant protein_state Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS |
| 14 21 Gly(-1) residue_name_number Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 60 69 histidine residue_name Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 93 97 (-2) residue_number Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 112 114 S2 site Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 130 139 S1 pocket site Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 164 184 antiparallel β-sheet structure_element Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS |
| 36 43 Gly(-1) residue_name_number Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). RESULTS |
| 95 99 Thr1 residue_name_number Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). RESULTS |
| 7 11 K81R mutant As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS |
| 45 56 active site site As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS |
| 58 62 Thr1 residue_name_number As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS |
| 65 70 Arg81 residue_name_number As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS |
| 98 108 propeptide structure_element As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS |
| 31 33 β5 protein Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 34 43 S1 pocket site Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 84 89 Met45 residue_name_number Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 128 135 His(-2) residue_name_number Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 143 150 S1 site site Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 201 210 autolysis ptm Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS |
| 61 65 Thr1 residue_name_number Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. RESULTS |
| 106 115 autolysis ptm Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. RESULTS |
| 12 22 eukaryotic taxonomy_domain Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS |
| 35 37 β5 protein Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS |
| 54 57 His residue_name Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS |
| 78 82 (-2) residue_number Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS |
| 90 100 propeptide structure_element Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS |
| 3 12 histidine residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 59 75 catalytic triads site As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 79 95 serine proteases protein_type As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 125 132 His(-2) residue_name_number As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 154 156 β5 protein As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 157 167 propeptide structure_element As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 171 188 exchanging it for experimental_method As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 189 192 Asn residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 194 197 Lys residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 199 202 Phe residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 207 210 Ala residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 216 221 yeast taxonomy_domain As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 303 309 H(-2)N mutant As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 314 320 H(-2)F mutant As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS |
| 56 62 H(-2)N mutant In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS |
| 67 73 H(-2)F mutant In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS |
| 74 80 mutant protein_state In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS |
| 81 85 yCPs complex_assembly In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS |
| 0 19 Structural analyses experimental_method Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS |
| 38 49 propeptides structure_element Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS |
| 57 63 mutant protein_state Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS |
| 64 68 yCPs complex_assembly Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS |
| 85 110 2FO–FC electron densities evidence Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS |
| 0 7 Gly(-1) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS |
| 12 15 Phe residue_name Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS |
| 16 23 Lys(-2) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS |
| 64 67 Ala residue_name Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS |
| 68 75 Asn(-2) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS |
| 40 49 processed protein_state This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS |
| 54 65 unprocessed protein_state This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS |
| 66 68 β5 protein This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS |
| 101 110 autolysis ptm This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS |
| 160 164 (-2) residue_number This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS |
| 40 44 (-2) residue_number Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 71 81 propeptide structure_element Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 85 114 creating mutants that combine experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 119 122 T1A mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 124 128 K81R mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 130 141 mutation(s) experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 147 153 H(-2)L mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 155 161 H(-2)T mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 165 171 H(-2)A mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 172 185 substitutions experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS |
| 0 7 Leu(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 26 31 yeast taxonomy_domain Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 32 34 β1 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 78 85 Thr(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 107 109 β2 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 110 121 propeptides structure_element Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 151 158 Ala(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 183 185 β5 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 186 195 S1 pocket site Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 239 244 Met45 residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS |
| 17 23 β5-T1A mutant As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). RESULTS |
| 37 43 yeasts taxonomy_domain As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). RESULTS |
| 14 32 crystal structures evidence We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS |
| 40 53 β5-H(-2)L-T1A mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS |
| 55 68 β5-H(-2)T-T1A mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS |
| 77 95 β5-H(-2)A-T1A-K81R mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS |
| 8 26 β5-H(-2)A-T1A-K81R mutant For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 54 61 Gly(-1) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 66 73 Ala(-2) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 111 118 Ala(-2) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 162 172 propeptide structure_element For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 180 205 substrate-binding channel site For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS |
| 17 28 prosegments structure_element By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 36 49 β5-H(-2)L-T1A mutant By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 58 71 β5-H(-2)T-T1A mutant By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 122 150 2FO–FC electron-density maps evidence By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 258 268 propeptide structure_element By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 277 284 His(-2) residue_name_number By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS |
| 19 26 Leu(-2) residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS |
| 31 38 Thr(-2) residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS |
| 64 85 S1 specificity pocket site Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS |
| 96 101 Met45 residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS |
| 48 55 His(-2) residue_name_number This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS |
| 63 65 β5 protein This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS |
| 66 76 propeptide structure_element This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS |
| 106 113 S1 site site This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS |
| 6 13 Gly(-1) residue_name_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 47 56 wild-type protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 58 60 WT protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 66 72 mutant protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 73 75 β5 protein Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 76 87 propeptides structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 179 183 Thr1 residue_name_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 240 244 (-2) residue_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 252 261 S1 pocket site Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 283 303 antiparallel β-sheet structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 321 330 autolysis ptm Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 338 348 propeptide structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS |
| 24 41 crystal structure evidence Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 47 55 chimeric protein_state Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 56 59 yCP complex_assembly Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 71 76 yeast taxonomy_domain Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 77 79 β1 protein Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 80 90 propeptide structure_element Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 91 102 replaced by experimental_method Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 107 109 β5 protein Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 110 121 counterpart structure_element Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS |
| 34 57 2FO–FC electron density evidence Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS |
| 65 67 β1 protein Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS |
| 68 79 active site site Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS |
| 36 43 Thr(-2) residue_name_number Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 47 49 β2 protein Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 51 58 Leu(-2) residue_name_number Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 70 72 β1 protein Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 76 89 not conserved protein_state Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 132 139 created experimental_method Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 142 151 β2-T(-2)V mutant Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 152 162 proteasome complex_assembly Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 163 169 mutant protein_state Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS |
| 17 23 β2-T1A mutant As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 24 42 crystal structures evidence As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 44 51 Thr(-2) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 52 66 hydrogen bonds bond_interaction As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 70 77 Gly(-1) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 131 144 β5-H(-2)T-T1A mutant As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 145 151 mutant protein_state As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 191 199 exchange experimental_method As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 203 210 Thr(-2) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 214 217 Val residue_name As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 221 223 β2 protein As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS |
| 13 40 2FO–FC electron-density map evidence Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS |
| 82 84 β2 protein Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS |
| 85 95 propeptide structure_element Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS |
| 127 133 β2-T1A mutant Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS |
| 134 144 proteasome complex_assembly Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS |
| 15 22 Val(-2) residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS |
| 45 52 S1 site site In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS |
| 57 64 Gly(-1) residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS |
| 176 180 Thr1 residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS |
| 62 82 active-site residues site These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS |
| 87 94 Gly(-1) residue_name_number These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS |
| 133 143 proteasome complex_assembly These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS |
| 4 15 active site site The active site of the proteasome RESULTS |
| 23 33 proteasome complex_assembly The active site of the proteasome RESULTS |
| 38 49 active site site Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS |
| 50 54 Thr1 residue_name_number Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS |
| 60 64 Thr1 residue_name_number Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS |
| 87 92 water chemical Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS |
| 16 24 immature protein_state However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS |
| 25 33 particle complex_assembly However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS |
| 34 38 Thr1 residue_name_number However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS |
| 60 70 propeptide structure_element However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS |
| 91 95 Thr1 residue_name_number However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS |
| 9 14 Lys33 residue_name_number Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS |
| 31 47 hydrogen-bonding bond_interaction Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS |
| 60 64 Thr1 residue_name_number Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS |
| 82 102 catalytically active protein_state Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS |
| 103 113 β subunits protein Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS |
| 11 27 catalytic tetrad site A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 44 48 Thr1 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 52 56 Thr1 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 61 66 Lys33 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 74 79 Asp17 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 109 114 water chemical A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 208 219 active site site A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS |
| 39 42 yCP complex_assembly Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS |
| 43 59 X-ray structures evidence Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS |
| 98 109 active site site Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS |
| 117 127 proteasome complex_assembly Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS |
| 0 8 Mutation experimental_method Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 12 14 β5 protein Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 15 20 Lys33 residue_name_number Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 24 27 Ala residue_name Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 82 117 structural and biochemical analyses experimental_method Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 162 181 propeptide cleavage ptm Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS |
| 21 28 β5-K33A mutant The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS |
| 29 35 mutant protein_state The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS |
| 77 88 β5-T1A-K81R mutant The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS |
| 89 94 yeast taxonomy_domain The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS |
| 70 77 L(-49)S mutant This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS |
| 85 87 β5 protein This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS |
| 88 98 propeptide structure_element This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS |
| 106 113 β5-K33A mutant This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS |
| 114 120 mutant protein_state This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS |
| 0 21 Structural comparison experimental_method Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 29 44 β5-L(-49)S-K33A mutant Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 49 60 β5-T1A-K81R mutant Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 61 73 active sites site Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 88 96 mutation experimental_method Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 100 105 Lys33 residue_name_number Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 109 112 Ala residue_name Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 150 154 Thr1 residue_name_number Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 171 181 propeptide structure_element Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS |
| 36 47 active-site site This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS |
| 98 100 β5 protein This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS |
| 101 112 active site site This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS |
| 41 46 Lys33 residue_name_number Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 69 76 β5-K33A mutant Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 77 83 mutant protein_state Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 94 104 propeptide structure_element Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 105 125 expressed separately experimental_method Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 149 151 pp chemical Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 152 157 trans protein_state Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS |
| 4 8 Thr1 residue_name_number The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS |
| 28 34 mutant protein_state The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS |
| 57 67 propeptide structure_element The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS |
| 26 43 crystal structure evidence Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 51 58 β5-K33A mutant Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 59 61 pp chemical Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 62 67 trans protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 68 74 mutant protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 75 90 in complex with protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 91 102 carfilzomib chemical Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 154 156 β5 protein Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 157 169 active sites site Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS |
| 9 20 acetylation ptm Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 28 32 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 65 72 β5-K33A mutant Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 73 75 pp chemical Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 76 81 trans protein_state Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 82 85 apo protein_state Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 86 103 crystal structure evidence Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 177 182 Lys33 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 199 203 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 235 239 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS |
| 17 34 crystal structure evidence Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 42 49 β5-K33A mutant Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 50 52 pp chemical Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 53 58 trans protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 59 65 mutant protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 66 83 without inhibitor protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 98 102 Thr1 residue_name_number Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 114 125 coordinates bond_interaction Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 141 146 water chemical Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS |
| 5 10 water chemical This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS |
| 11 25 hydrogen bonds bond_interaction This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS |
| 34 39 Arg19 residue_name_number This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS |
| 54 59 Asp17 residue_name_number This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS |
| 128 134 mutant protein_state This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS |
| 73 78 Lys33 residue_name_number Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 89 91 WT protein_state Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 92 102 proteasome complex_assembly Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 103 112 structure evidence Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 168 173 Lys33 residue_name_number Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 198 207 autolysis ptm Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS |
| 0 25 Conservative substitution experimental_method Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 29 34 Lys33 residue_name_number Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 38 41 Arg residue_name Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 49 58 autolysis ptm Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 66 68 β5 protein Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 91 96 yeast taxonomy_domain Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS |
| 6 10 Thr1 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 44 46 WT protein_state While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 47 51 yCPs complex_assembly While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 53 58 Arg33 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 72 85 hydrogen bond bond_interaction While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 89 94 Asp17 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 121 123 β5 protein While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 124 135 active site site While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS |
| 4 25 conservative mutation experimental_method The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS |
| 29 34 Asp17 residue_name_number The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS |
| 38 41 Asn residue_name The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS |
| 53 55 β5 protein The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS |
| 63 66 yCP complex_assembly The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS |
| 49 56 L(-49)S mutant Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 72 74 β5 protein Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 75 85 propeptide structure_element Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 124 131 β5-D17N mutant Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 132 138 mutant protein_state Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 139 142 yCP complex_assembly Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS |
| 17 42 crystallographic analysis experimental_method As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 49 55 mutant protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 56 58 β5 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 71 90 partially processed protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 147 157 proteasome complex_assembly As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 168 179 carfilzomib chemical As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 207 209 β1 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 214 216 β2 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 227 229 WT protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 230 232 β5 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS |
| 19 22 cis protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 34 44 expression experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 52 54 β5 protein In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 55 65 propeptide structure_element In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 69 74 trans protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 99 108 isolation experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 113 128 crystallization experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 136 140 D17N mutant In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 141 147 mutant protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 148 158 proteasome complex_assembly In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS |
| 26 33 β5-D17N mutant The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 34 36 pp chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 40 45 trans protein_state The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 46 48 CP complex_assembly The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 71 73 β5 protein The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 91 142 N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 144 156 Suc-LLVY-AMC chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 162 205 carboxybenzyl-Gly-Gly-Leu-para-nitroanilide chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 207 216 Z-GGL-pNA chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 280 285 Asp17 residue_name_number The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 349 355 mature protein_state The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 356 366 proteasome complex_assembly The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS |
| 16 23 β5-D17N mutant Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 24 26 pp chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 27 32 trans protein_state Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 33 36 yCP complex_assembly Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 37 54 crystal structure evidence Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 81 83 WT protein_state Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 84 87 yCP complex_assembly Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 117 137 co-crystal structure evidence Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 147 165 α′, β′ epoxyketone chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 176 187 carfilzomib chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 243 245 β5 protein Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 246 257 active site site Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS |
| 69 71 β5 protein This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 72 76 Thr1 residue_name_number This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 85 102 crystal structure evidence This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 110 117 β5-D17N mutant This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 118 120 pp chemical This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 121 124 cis protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 125 131 mutant protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 132 147 in complex with protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 148 159 carfilzomib chemical This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS |
| 0 9 Autolysis ptm Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS |
| 49 56 β5-D17N mutant Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS |
| 106 111 Asn17 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS |
| 163 168 Lys33 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS |
| 191 195 Thr1 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS |
| 17 21 E17A mutant In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 22 28 mutant protein_state In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 48 57 β-subunit protein In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 65 73 archaeon taxonomy_domain In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 74 98 Thermoplasma acidophilum species In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 108 117 autolysis ptm In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS |
| 25 35 X-ray data evidence Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 43 50 β5-D17N mutant Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 51 57 mutant protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 67 77 propeptide structure_element Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 78 87 expressed experimental_method Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 91 94 cis protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 102 107 trans protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS |
| 47 50 CPs complex_assembly On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 82 97 catalytic triad site On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 112 116 Thr1 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 118 123 Lys33 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 128 131 Asp residue_name On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 132 137 Glu17 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 147 181 autocatalytic precursor processing ptm On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS |
| 60 65 water chemical This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 94 100 mature protein_state This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 101 103 WT protein_state This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 116 127 active site site This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 163 167 Thr1 residue_name_number This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 173 177 Thr1 residue_name_number This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS |
| 16 27 active-site site To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 46 69 exchanged the conserved experimental_method To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 70 76 Asp166 residue_name_number To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 89 92 Asn residue_name To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 100 105 yeast taxonomy_domain To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 106 108 β5 protein To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS |
| 0 6 Asp166 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS |
| 12 27 hydrogen-bonded bond_interaction Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS |
| 31 35 Thr1 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS |
| 43 49 Ser129 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS |
| 56 62 Ser169 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS |
| 4 12 β5-D166N mutant The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS |
| 13 15 pp chemical The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS |
| 16 19 cis protein_state The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS |
| 20 25 yeast taxonomy_domain The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS |
| 26 32 mutant protein_state The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS |
| 0 10 X-ray data evidence X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 18 26 β5-D166N mutant X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 27 33 mutant protein_state X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 52 54 β5 protein X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 55 65 propeptide structure_element X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 109 115 Ser129 residue_name_number X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 140 146 Asn166 residue_name_number X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS |
| 11 16 water chemical Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS |
| 29 37 bound to protein_state Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS |
| 38 44 Ser129 residue_name_number Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS |
| 51 55 Thr1 residue_name_number Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS |
| 101 121 precursor processing ptm Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS |
| 4 18 hydrogen bonds bond_interaction The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. RESULTS |
| 29 35 Ser169 residue_name_number The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. RESULTS |
| 0 7 Soaking experimental_method Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 12 20 β5-D166N mutant Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 21 29 crystals experimental_method Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 35 46 carfilzomib chemical Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 51 56 MG132 chemical Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 94 98 Thr1 residue_name_number Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS |
| 7 26 carfilzomib complex complex_assembly In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 27 36 structure evidence In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 38 42 Thr1 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 49 53 Thr1 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 104 110 Ser129 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 122 124 WT protein_state In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS |
| 7 24 MG132-bound state protein_state In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 26 30 Thr1 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 35 45 unmodified protein_state In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 73 79 Ser129 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 83 98 hydrogen-bonded bond_interaction In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 104 109 water chemical In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 130 136 Asn166 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS |
| 8 11 Asn residue_name Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS |
| 39 45 Asp166 residue_name_number Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS |
| 91 94 Ala residue_name Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS |
| 138 147 autolysis ptm Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS |
| 27 33 Asp166 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS |
| 38 44 Ser129 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS |
| 110 114 Thr1 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS |
| 123 132 autolysis ptm These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS |
| 0 12 Substitution experimental_method Substitution of the active-site Thr1 by Cys RESULTS |
| 20 31 active-site site Substitution of the active-site Thr1 by Cys RESULTS |
| 32 36 Thr1 residue_name_number Substitution of the active-site Thr1 by Cys RESULTS |
| 40 43 Cys residue_name Substitution of the active-site Thr1 by Cys RESULTS |
| 0 8 Mutation experimental_method Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 12 16 Thr1 residue_name_number Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 20 23 Cys residue_name Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 40 54 20S proteasome complex_assembly Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 64 72 archaeon taxonomy_domain Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 73 87 T. acidophilum species Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS |
| 3 8 yeast taxonomy_domain In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS |
| 15 23 mutation experimental_method In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS |
| 90 100 propeptide structure_element In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS |
| 137 152 X-ray structure evidence In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS |
| 18 20 β5 protein In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS |
| 53 60 cleaved protein_state In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS |
| 61 71 propeptide structure_element In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS |
| 72 83 still bound protein_state In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS |
| 91 116 substrate-binding channel site In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS |
| 0 7 His(-2) residue_name_number His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 21 30 S2 pocket site His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 53 64 β5-T1A-K81R mutant His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 65 71 mutant protein_state His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 108 118 propeptide structure_element His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 126 129 T1C mutant His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 130 136 mutant protein_state His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 147 167 antiparallel β-sheet structure_element His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 211 216 MG132 chemical His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS |
| 37 40 T1C mutant On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 41 47 mutant protein_state On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 56 66 propeptide structure_element On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 93 104 active site site On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 122 131 autolysis ptm On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 183 198 crystallization experimental_method On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS |
| 42 44 β5 protein Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS |
| 65 67 CP complex_assembly Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS |
| 107 117 propeptide structure_element Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS |
| 8 29 propeptide hydrolysis ptm Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS |
| 35 41 β5-T1C mutant Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS |
| 42 53 active site site Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS |
| 57 79 catalytically inactive protein_state Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS |
| 14 30 soaking crystals experimental_method In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 40 42 CP complex_assembly In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 54 64 bortezomib chemical In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 68 79 carfilzomib chemical In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 98 100 β1 protein In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 105 107 β2 protein In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 108 120 active sites site In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 140 146 β5-T1C mutant In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 147 166 proteolytic centres site In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 167 177 unmodified protein_state In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 230 237 cleaved protein_state In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 238 248 propeptide structure_element In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS |
| 14 29 structural data evidence Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS |
| 61 65 Cys1 residue_name_number Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS |
| 127 131 Thr1 residue_name_number Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS |
| 18 31 hydrogen bond bond_interaction Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS |
| 73 78 Lys33 residue_name_number Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS |
| 82 84 WT protein_state Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS |
| 85 88 CPs complex_assembly Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS |
| 104 108 Cys1 residue_name_number Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS |
| 13 40 2FO–FC electron-density map evidence Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS |
| 48 51 T1C mutant Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS |
| 52 58 mutant protein_state Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS |
| 79 84 Lys33 residue_name_number Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS |
| 91 101 disordered protein_state Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS |
| 90 95 Lys33 residue_name_number Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. RESULTS |
| 99 113 hydrogen bonds bond_interaction Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. RESULTS |
| 15 18 Thr residue_name The benefit of Thr over Ser as the active-site nucleophile RESULTS |
| 24 27 Ser residue_name The benefit of Thr over Ser as the active-site nucleophile RESULTS |
| 4 15 proteasomes complex_assembly All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS |
| 16 31 strictly employ protein_state All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS |
| 32 41 threonine residue_name All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS |
| 49 68 active-site residue site All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS |
| 80 86 serine residue_name All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS |
| 62 68 β5-T1S mutant To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). RESULTS |
| 69 75 mutant protein_state To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). RESULTS |
| 0 15 Activity assays experimental_method Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 25 27 β5 protein Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 47 59 Suc-LLVY-AMC chemical Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 104 107 T1S mutant Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 108 114 mutant protein_state Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 150 152 WT protein_state Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 153 164 proteasomes complex_assembly Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS |
| 39 48 Z-GGL-pNA chemical By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a). RESULTS |
| 0 17 Crystal structure evidence Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 34 40 β5-T1S mutant Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 41 47 mutant protein_state Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 58 78 precursor processing ptm Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 94 108 ligand-complex complex_assembly Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 109 119 structures evidence Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 125 135 bortezomib chemical Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 140 151 carfilzomib chemical Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 197 201 Ser1 residue_name_number Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS |
| 13 16 apo protein_state However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS |
| 17 34 crystal structure evidence However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS |
| 49 53 Ser1 residue_name_number However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS |
| 80 105 substrate-binding channel site However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS |
| 14 18 Thr1 residue_name_number Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS |
| 24 26 WT protein_state Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS |
| 27 29 CP complex_assembly Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS |
| 30 40 structures evidence Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS |
| 42 46 Ser1 residue_name_number Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS |
| 30 34 Ser1 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS |
| 41 56 hydrogen-bonded bond_interaction Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS |
| 60 65 Lys33 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS |
| 179 183 Thr1 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS |
| 4 23 active-site residue site The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS |
| 24 28 Thr1 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS |
| 89 113 hydrophobic interactions bond_interaction The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS |
| 119 123 Thr3 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS |
| 128 133 Ala46 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS |
| 36 40 Thr1 residue_name_number Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. RESULTS |
| 139 143 Ser1 residue_name_number Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. RESULTS |
| 62 65 T1S mutant In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). RESULTS |
| 66 72 mutant protein_state In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). RESULTS |
| 14 20 mutant protein_state In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS |
| 21 31 proteasome complex_assembly In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS |
| 55 65 proteasome complex_assembly In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS |
| 80 90 bortezomib chemical In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS |
| 106 117 carfilzomib chemical In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS |
| 14 31 inhibitor complex complex_assembly Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 32 42 structures evidence Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 94 96 WT protein_state Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 97 100 yCP complex_assembly Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 101 111 structures evidence Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 113 137 with the same inhibitors protein_state Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS |
| 13 21 affinity evidence Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS |
| 42 53 carfilzomib chemical Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS |
| 106 131 substrate-binding channel site Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS |
| 151 161 bortezomib chemical Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS |
| 11 30 mean residence time evidence Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS |
| 34 45 carfilzomib chemical Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS |
| 53 64 active site site Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS |
| 123 127 Ser1 residue_name_number Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS |
| 89 98 threonine residue_name Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS |
| 133 144 proteasomes complex_assembly Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS |
| 189 214 proteasome-like proteases protein_type Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS |
| 223 227 HslV protein Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS |
| 4 18 20S proteasome complex_assembly The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS |
| 19 21 CP complex_assembly The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS |
| 35 57 non-lysosomal protease protein_type The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS |
| 61 71 eukaryotic taxonomy_domain The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS |
| 4 13 β-subunit protein The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS |
| 14 25 propeptides structure_element The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS |
| 48 50 β5 protein The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS |
| 107 109 CP complex_assembly The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS |
| 59 63 Thr1 residue_name_number In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. DISCUSS |
| 78 91 N-acetylation ptm In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. DISCUSS |
| 17 28 prosegments structure_element By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 32 42 β subunits protein By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 63 71 archaeal taxonomy_domain By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 72 82 proteasome complex_assembly By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 107 131 heterologously expressed experimental_method By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 135 151 Escherichia coli species By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS |
| 3 13 eukaryotes taxonomy_domain In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS |
| 52 54 β1 protein In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS |
| 59 61 β2 protein In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS |
| 62 73 propeptides structure_element In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS |
| 9 19 removal of experimental_method However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS |
| 24 26 β5 protein However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS |
| 27 37 prosegment structure_element However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS |
| 71 73 β5 protein These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 74 84 propeptide structure_element These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 100 102 β5 protein These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 103 114 active site site These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 150 160 eukaryotic taxonomy_domain These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 161 163 CP complex_assembly These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS |
| 27 44 atomic structures evidence Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS |
| 56 62 β5-T1A mutant Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS |
| 135 137 β5 protein Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS |
| 138 148 propeptide structure_element Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS |
| 17 21 (-2) residue_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 50 60 propeptide structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 80 87 Gly(-1) residue_name_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 98 108 structures evidence Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 158 162 Thr1 residue_name_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 197 207 tight turn structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 225 235 prosegment structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 247 249 β1 protein Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS |
| 57 64 Gly(-1) residue_name_number From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS |
| 69 73 Thr1 residue_name_number From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS |
| 118 129 active site site From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS |
| 147 156 autolysis ptm From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS |
| 30 43 N-acetylation ptm In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS |
| 51 55 Thr1 residue_name_number In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS |
| 88 92 Thr1 residue_name_number In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS |
| 4 14 propeptide structure_element The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS |
| 43 68 substrate-binding channel site The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS |
| 90 97 Gly(-1) residue_name_number The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS |
| 162 166 (-2) residue_number The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS |
| 28 30 CP complex_assembly Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. DISCUSS |
| 112 131 propeptide cleavage ptm Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. DISCUSS |
| 29 38 CP:ligand complex_assembly On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS |
| 146 156 proteasome complex_assembly On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS |
| 157 181 active-site architecture site On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS |
| 13 28 catalytic triad site We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 37 48 active site site We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 56 58 CP complex_assembly We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 82 86 Thr1 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 88 93 Lys33 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 98 101 Asp residue_name We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 102 107 Glu17 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 162 172 eukaryotic taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 174 183 bacterial taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 188 196 archaeal taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 197 207 proteasome complex_assembly We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS |
| 0 5 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 58 79 autocatalytic removal ptm Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 87 98 propeptides structure_element Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 147 152 Asp17 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 163 168 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 231 244 hydrogen bond bond_interaction Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 255 260 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 265 270 Asp17 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS |
| 19 29 proteasome complex_assembly Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS |
| 33 50 Thr–Lys–Asp triad site Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS |
| 68 82 L-asparaginase protein_type Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS |
| 107 110 Lys residue_name Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst. DISCUSS |
| 36 47 active site site In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS |
| 72 76 Thr1 residue_name_number In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS |
| 90 104 hydrogen bonds bond_interaction In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS |
| 207 231 endoproteolytic cleavage ptm In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS |
| 235 239 Thr1 residue_name_number In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS |
| 51 55 Thr1 residue_name_number Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 81 95 hydrogen bonds bond_interaction Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 127 140 fellutamide B chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 149 161 α-ketoamides chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 163 179 homobelactosin C chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 191 208 salinosporamide A chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS |
| 47 56 omuralide chemical Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS |
| 60 64 Thr1 residue_name_number Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS |
| 123 127 Thr1 residue_name_number Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS |
| 24 28 Thr1 residue_name_number The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. DISCUSS |
| 35 51 hydrogen-bridged bond_interaction The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. DISCUSS |
| 14 25 acetylation ptm In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. DISCUSS |
| 33 37 Thr1 residue_name_number In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. DISCUSS |
| 50 54 Thr1 residue_name_number By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). DISCUSS |
| 98 108 proteasome complex_assembly Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. DISCUSS |
| 114 118 Thr1 residue_name_number Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. DISCUSS |
| 13 17 Thr1 residue_name_number Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. DISCUSS |
| 226 230 Thr1 residue_name_number Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. DISCUSS |
| 7 16 autolysis ptm During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. DISCUSS |
| 21 25 Thr1 residue_name_number During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. DISCUSS |
| 60 64 Thr1 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS |
| 98 115 aspartic acid 166 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS |
| 120 126 Ser129 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS |
| 138 142 Thr1 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS |
| 13 19 Ser129 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS |
| 24 30 Asp166 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS |
| 73 77 Thr1 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS |
| 67 75 mutation experimental_method Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 76 81 D166A mutant Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 91 100 autolysis ptm Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 108 116 archaeal taxonomy_domain Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 117 119 CP complex_assembly Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 128 136 exchange experimental_method Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 137 142 D166N mutant Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 177 182 yeast taxonomy_domain Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 183 185 CP complex_assembly Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS |
| 4 12 mutation experimental_method The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS |
| 13 18 D166N mutant The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS |
| 37 41 Thr1 residue_name_number The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS |
| 116 120 Thr1 residue_name_number The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS |
| 79 87 β5-D166N mutant This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS |
| 88 94 mutant protein_state This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS |
| 150 161 carfilzomib chemical This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS |
| 11 21 proteasome complex_assembly Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. DISCUSS |
| 48 60 second triad site Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. DISCUSS |
| 6 11 Lys33 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 19 24 Asp17 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 59 63 Thr1 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 85 91 Ser129 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 98 104 Asp166 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 156 160 Thr1 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS |
| 28 32 Thr1 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 33 38 Lys33 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 39 44 Asp17 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 45 60 catalytic triad site In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 62 83 crystallographic data evidence In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 91 115 proteolytically inactive protein_state In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 116 122 β5-T1C mutant In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 123 129 mutant protein_state In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 166 171 Lys33 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 179 183 Cys1 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS |
| 18 21 Cys residue_name However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS |
| 54 64 propeptide structure_element However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS |
| 78 85 cleaved protein_state However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS |
| 48 57 autolysis ptm While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. DISCUSS |
| 16 29 Ntn hydrolase protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 30 48 penicillin acylase protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 50 62 substitution experimental_method Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 70 79 catalytic protein_state Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 91 94 Ser residue_name Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 106 109 Cys residue_name Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 115 126 inactivates protein_state Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 131 137 enzyme protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 156 176 precursor processing ptm Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS |
| 23 25 CP complex_assembly To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 47 56 threonine residue_name To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 64 83 active-site residue site To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 95 101 β5-T1S mutant To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 102 108 mutant protein_state To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 116 119 yCP complex_assembly To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 141 171 biochemically and structurally experimental_method To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS |
| 0 15 Activity assays experimental_method Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS |
| 25 31 β5-T1S mutant Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS |
| 32 38 mutant protein_state Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS |
| 68 80 Suc-LLVY-AMC chemical Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS |
| 48 54 β5-T1S mutant We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 55 61 mutant protein_state We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 62 65 yCP complex_assembly We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 112 122 proteasome complex_assembly We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 134 144 bortezomib chemical We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 149 160 carfilzomib chemical We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS |
| 0 19 Structural analyses evidence Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 52 55 T1S mutant Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 56 62 mutant protein_state Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 98 111 strict use of protein_state Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 112 115 Thr residue_name Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 128 139 proteasomes complex_assembly Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS |
| 0 4 Thr1 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 29 40 active site site Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 44 68 hydrophobic interactions bond_interaction Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 97 102 Ala46 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 109 114 Lys33 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 139 143 Thr3 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS |
| 9 31 proteolytically active protein_state Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 32 42 proteasome complex_assembly Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 57 64 archaea taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 66 71 yeast taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 76 83 mammals taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 161 164 Thr residue_name Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 168 171 Ile residue_name Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 184 185 3 residue_number Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 267 271 Thr1 residue_name_number Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS |
| 15 19 Thr1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS |
| 43 47 Ser1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS |
| 77 81 Thr1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS |
| 107 109 WT protein_state In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS |
| 110 116 enzyme complex_assembly In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS |
| 16 35 threonine aspartase protein_type Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 36 44 Taspase1 protein Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 46 54 mutation experimental_method Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 62 73 active-site site Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 74 80 Thr234 residue_name_number Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 84 87 Ser residue_name Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 154 160 Thr234 residue_name_number Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 168 170 WT protein_state Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS |
| 24 30 serine residue_name Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 31 37 mutant protein_state Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 41 47 active protein_state Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 49 58 threonine residue_name Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 99 109 proteasome complex_assembly Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 110 121 active site site Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS |
| 27 36 threonine residue_name The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 45 55 proteasome complex_assembly The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 56 67 active site site The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 172 184 conservation protein_state The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 192 196 Thr1 residue_name_number The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 212 223 proteasomes complex_assembly The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 229 237 bacteria taxonomy_domain The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 241 251 eukaryotes taxonomy_domain The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS |
| 28 39 propeptides structure_element Conformation of proteasomal propeptides. FIG |
| 4 28 Structural superposition experimental_method (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 36 42 β1-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 43 53 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 62 69 matured protein_state (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 70 72 WT protein_state (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 73 75 β1 protein (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 76 87 active-site site (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 88 92 Thr1 residue_name_number (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG |
| 18 30 (-5) to (-1) residue_range Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG |
| 38 44 β1-T1A mutant Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG |
| 45 55 propeptide structure_element Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG |
| 29 50 S1 specificity pocket site The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG |
| 60 62 45 residue_number The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG |
| 29 50 S1 specificity pocket site The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG |
| 60 62 45 residue_number The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG |
| 31 38 Gly(-1) residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 43 47 Ala1 residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 55 65 propeptide structure_element Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 75 80 G(-1) residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 133 142 processed protein_state Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 143 145 WT protein_state Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 146 157 active-site site Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 158 162 Thr1 residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG |
| 40 44 Thr1 residue_name_number The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). FIG |
| 80 87 Gly(-1) residue_name_number The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). FIG |
| 4 28 Structural superposition experimental_method (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 36 42 β1-T1A mutant (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 43 53 propeptide structure_element (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 62 68 β2-T1A mutant (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 69 79 propeptide structure_element (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 155 159 Ala1 residue_name_number (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 164 171 Gly(-1) residue_name_number (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG |
| 0 7 Thr(-2) residue_name_number Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG |
| 13 28 hydrogen-bonded bond_interaction Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG |
| 32 39 Gly(-1) residue_name_number Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG |
| 4 28 Structural superposition experimental_method (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG |
| 36 42 β1-T1A mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG |
| 48 54 β2-T1A mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG |
| 63 74 β5-T1A-K81R mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG |
| 75 85 propeptide structure_element (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG |
| 14 18 (-2) residue_number While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 26 28 β1 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 33 35 β2 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 36 47 prosegments structure_element While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 56 65 S1 pocket site While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 67 74 His(-2) residue_name_number While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 82 84 β5 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 85 95 propeptide structure_element While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 109 118 S2 pocket site While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG |
| 56 63 Gly(-1) residue_name_number Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. FIG |
| 113 117 Thr1 residue_name_number Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. FIG |
| 4 17 hydrogen bond bond_interaction The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG |
| 26 33 Thr(-2) residue_name_number The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG |
| 40 47 Gly(-1) residue_name_number The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG |
| 0 9 Mutations experimental_method Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG |
| 21 25 (-2) residue_number Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG |
| 49 59 propeptide structure_element Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG |
| 77 86 autolysis ptm Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG |
| 4 28 Structural superposition experimental_method (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 36 42 β1-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 43 53 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 62 75 β5-H(-2)L-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 76 82 mutant protein_state (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 83 93 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG |
| 4 8 (-2) residue_number The (-2) residues of both prosegments point into the S1 pocket. FIG |
| 26 37 prosegments structure_element The (-2) residues of both prosegments point into the S1 pocket. FIG |
| 53 62 S1 pocket site The (-2) residues of both prosegments point into the S1 pocket. FIG |
| 4 28 Structural superposition experimental_method (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 36 38 β5 protein (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 39 50 propeptides structure_element (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 58 71 β5-H(-2)L-T1A mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 73 86 β5-H(-2)T-T1A mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 88 106 β5-(H-2)A-T1A-K81R mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 111 122 β5-T1A-K81R mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 123 129 mutant protein_state (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 130 141 proteasomes complex_assembly (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG |
| 19 31 (-2) to (-4) residue_range While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG |
| 60 67 Gly(-1) residue_name_number While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG |
| 72 76 Ala1 residue_name_number While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG |
| 96 106 structures evidence While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG |
| 4 28 Structural superposition experimental_method (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 36 42 β2-T1A mutant (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 43 53 propeptide structure_element (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 62 75 β5-H(-2)T-T1A mutant (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 76 82 mutant protein_state (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 83 93 propeptide structure_element (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG |
| 4 8 (-2) residue_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 26 37 prosegments structure_element The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 53 62 S1 pocket site The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 73 80 Thr(-2) residue_name_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 86 88 β2 protein The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 97 112 hydrogen bridge bond_interaction The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 116 123 Gly(-1) residue_name_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG |
| 4 28 Structural superposition experimental_method (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 36 43 matured protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 44 46 β2 protein (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 47 58 active site site (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 64 66 WT protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 67 73 β2-T1A mutant (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 74 84 propeptide structure_element (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 93 102 β2-T(-2)V mutant (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 103 109 mutant protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 110 120 propeptide structure_element (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG |
| 9 16 Val(-2) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG |
| 51 60 S1 pocket site Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG |
| 98 105 Gly(-1) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG |
| 144 148 Thr1 residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG |
| 182 189 Gly(-1) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG |
| 64 75 active site site Architecture and proposed reaction mechanism of the proteasomal active site. FIG |
| 4 28 Hydrogen-bonding network site (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG |
| 36 42 mature protein_state (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG |
| 43 45 WT protein_state (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG |
| 46 48 β5 protein (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG |
| 61 72 active site site (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG |
| 0 4 Thr1 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG |
| 10 25 hydrogen-bonded bond_interaction Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG |
| 29 34 Lys33 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG |
| 76 81 Asp17 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG |
| 4 8 Thr1 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 34 48 hydrogen bonds bond_interaction The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 54 60 Ser129 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 95 98 168 residue_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 100 106 Ser169 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 113 119 Asp166 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 151 171 active-site residues site The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 184 200 hydrogen bonding bond_interaction The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 205 223 strictly conserved protein_state The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 232 250 proteolytic centre site The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 264 277 superposition experimental_method The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 285 295 β subunits protein The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG |
| 4 28 Structural superposition experimental_method (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 36 38 WT protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 39 41 β5 protein (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 50 57 β5-K33A mutant (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 58 60 pp chemical (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 61 66 trans protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 67 73 mutant protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 74 85 active site site (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG |
| 17 22 water chemical In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG |
| 83 85 WT protein_state In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG |
| 126 131 Lys33 residue_name_number In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG |
| 13 18 Lys33 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 24 29 water chemical Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 39 53 hydrogen bonds bond_interaction Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 57 62 Arg19 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 65 70 Asp17 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 77 81 Thr1 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG |
| 38 43 water chemical Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG |
| 77 81 Thr1 residue_name_number Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG |
| 99 119 active-site residues site Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG |
| 45 79 autocatalytic precursor processing ptm (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. FIG |
| 103 113 proteasome complex_assembly (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. FIG |
| 4 15 active-site site The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG |
| 16 20 Thr1 residue_name_number The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG |
| 46 56 propeptide structure_element The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG |
| 0 9 Autolysis ptm Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG |
| 68 72 Thr1 residue_name_number Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG |
| 79 84 Lys33 residue_name_number Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG |
| 4 22 strictly conserved protein_state The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. FIG |
| 37 42 Gly47 residue_name_number The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. FIG |
| 43 47 Thr1 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG |
| 102 112 propeptide structure_element Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG |
| 151 157 Asp166 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG |
| 164 170 Ser129 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG |
| 6 10 Thr1 residue_name_number Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. FIG |
| 26 31 water chemical Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. FIG |
| 33 44 active-site site On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). FIG |
| 45 49 Thr1 residue_name_number On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). FIG |
| 12 16 Thr1 residue_name_number The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. FIG |
| 27 31 Thr1 residue_name_number The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. FIG |
| 55 60 water chemical The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. FIG |
| 4 14 proteasome complex_assembly The proteasome favours threonine as the active-site nucleophile. FIG |
| 23 32 threonine residue_name The proteasome favours threonine as the active-site nucleophile. FIG |
| 4 35 Growth tests by serial dilution experimental_method (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 39 41 WT protein_state (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 52 54 β5 protein (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 56 62 mutant protein_state (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 63 68 yeast taxonomy_domain (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 107 118 active-site site (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 119 126 mutants experimental_method (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG |
| 13 15 WT protein_state (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG |
| 20 26 mutant protein_state (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG |
| 27 38 proteasomes complex_assembly (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG |
| 89 91 β5 protein (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG |
| 113 125 Suc-LLVY-AMC chemical (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG |
| 24 51 2FO–FC electron-density map evidence (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG |
| 88 94 β5-T1C mutant (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG |
| 95 105 propeptide structure_element (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG |
| 4 14 prosegment structure_element The prosegment is cleaved but still bound in the substrate-binding channel. FIG |
| 18 25 cleaved protein_state The prosegment is cleaved but still bound in the substrate-binding channel. FIG |
| 30 41 still bound protein_state The prosegment is cleaved but still bound in the substrate-binding channel. FIG |
| 49 74 substrate-binding channel site The prosegment is cleaved but still bound in the substrate-binding channel. FIG |
| 9 16 His(-2) residue_name_number Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG |
| 37 46 S1 pocket site Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG |
| 57 62 Met45 residue_name_number Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG |
| 101 112 β5-T1A-K81R mutant Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG |
| 113 119 mutant protein_state Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG |
| 4 28 Structural superposition experimental_method (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 36 47 β5-T1A-K81R mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 56 62 β5-T1C mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 63 69 mutant protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 88 90 WT protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 91 93 β5 protein (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 107 131 Structural superposition experimental_method (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 139 145 β5-T1C mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 146 156 propeptide structure_element (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 166 172 β1-T1A mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 173 184 active site site (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 200 202 WT protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 203 205 β5 protein (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 206 217 active site site (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 218 233 in complex with protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 238 248 proteasome complex_assembly (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 259 264 MG132 chemical (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG |
| 4 13 inhibitor chemical The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG |
| 29 40 propeptides structure_element The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG |
| 76 101 substrate-binding channel site The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG |
| 4 28 Structural superposition experimental_method (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 36 38 WT protein_state (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 39 41 β5 protein (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 46 52 β5-T1C mutant (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 53 59 mutant protein_state (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 60 72 active sites site (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 137 141 Thr1 residue_name_number (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 170 174 Cys1 residue_name_number (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG |
| 4 28 Structural superposition experimental_method (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 36 38 WT protein_state (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 39 41 β5 protein (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 46 52 β5-T1S mutant (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 53 59 mutant protein_state (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 60 72 active sites site (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 130 134 Thr1 residue_name_number (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 139 143 Ser1 residue_name_number (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG |
| 4 31 2FO–FC electron-density map evidence The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. FIG |
| 36 40 Ser1 residue_name_number The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. FIG |
| 24 28 Thr1 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG |
| 44 68 hydrophobic interactions bond_interaction (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG |
| 74 79 Ala46 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG |
| 86 90 Thr3 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG |
| 0 4 Ser1 residue_name_number Ser1 lacks this stabilization and is therefore rotated by 60°. FIG |
| 5 10 lacks protein_state Ser1 lacks this stabilization and is therefore rotated by 60°. FIG |
| 14 16 WT protein_state Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 21 27 mutant protein_state Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 28 34 β5-T1S mutant Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 35 46 proteasomes complex_assembly Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 50 60 bortezomib chemical Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 65 76 carfilzomib chemical Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG |
| 0 17 Inhibition assays experimental_method Inhibition assays (left panel). FIG |
| 9 14 yeast taxonomy_domain Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 15 26 proteasomes complex_assembly Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 78 80 β5 protein Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 108 118 bortezomib chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 123 134 carfilzomib chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 155 167 Suc-LLVY-AMC chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG |
| 0 11 IC50 values evidence IC50 values were determined in triplicate; s.d.'s are indicated by error bars. FIG |
| 10 21 IC50 values evidence Note that IC50 values depend on time and enzyme concentration. FIG |
| 0 11 Proteasomes complex_assembly Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). FIG |
| 0 10 Structures evidence Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG |
| 18 24 β5-T1S mutant Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG |
| 25 31 mutant protein_state Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG |
| 35 60 complex with both ligands complex_assembly Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG |
| 93 97 Ser1 residue_name_number Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG |
| 4 32 2FO–FC electron-density maps evidence The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG |
| 49 53 Ser1 residue_name_number The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG |
| 112 119 P1 site site The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG |
| 121 125 Leu1 residue_name_number The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG |
| 4 6 WT protein_state The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 7 35 proteasome:inhibitor complex complex_assembly The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 36 46 structures evidence The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 67 71 Thr1 residue_name_number The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 86 98 superimposed experimental_method The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 120 128 mutation experimental_method The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 132 136 Thr1 residue_name_number The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 140 143 Ser residue_name The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 180 190 bortezomib chemical The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
| 194 205 carfilzomib chemical The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG |
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