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