PMC 20140719 pmc.key 4792962 CC BY no 0 0 10.1038/ncomms10900 ncomms10900 4792962 26964885 10900 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:Huber;given-names:Eva M. surname:Heinemeyer;given-names:Wolfgang surname:Li;given-names:Xia surname:Arendt;given-names:Cassandra S. surname:Hochstrasser;given-names:Mark surname:Groll;given-names:Michael TITLE front 7 2016 0 A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome ptm MESH: melaniev@ebi.ac.uk 2023-03-20T11:54:48Z autocatalytic activation complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T11:54:41Z 20S proteasome ABSTRACT abstract 87 Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. 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. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. 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. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T11:54:41Z 20S proteasome structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:28Z active-site residue_name SO: melaniev@ebi.ac.uk 2023-03-20T11:59:33Z threonines ptm MESH: melaniev@ebi.ac.uk 2023-03-22T10:33:38Z autocatalytically protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T11:59:38Z strict conservation residue_name SO: melaniev@ebi.ac.uk 2023-03-20T11:59:41Z threonine experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T12:00:07Z mutagenesis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T12:00:09Z X-ray crystallography experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T12:00:12Z biochemical assays residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:18Z Lys33 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:37Z Asp17 site SO: melaniev@ebi.ac.uk 2023-03-20T12:00:53Z catalytic triad experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T12:00:57Z Substitution residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:07Z inactivates complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:22Z proteasome mutant MESH: melaniev@ebi.ac.uk 2023-03-20T12:01:44Z Thr1Ser protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:59Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:02:10Z wild type residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:04:54Z Ser1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T12:02:47Z propeptide autolysis complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:22Z proteasome protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T12:02:54Z threonine protease ABSTRACT abstract 1120 The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:22Z proteasome protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T12:02:54Z threonine protease complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:22Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:13Z active site INTRO paragraph 1415 The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. 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. While the inactive α subunits build the two outer rings, the β subunits form the inner rings. 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. 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). Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:13:51Z 20S proteasome core particle complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T13:14:09Z non-lysosomal protease taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:20Z α protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:15:38Z heptameric structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:15:45Z rings structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:15:49Z hollow cylinder protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:15:56Z inactive protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:05Z α subunits structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:15:45Z rings protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:15:45Z rings protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:42Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:17:20Z proteolytic active centres complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:44Z prosegments ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:17:55Z autocatalytically removed site SO: melaniev@ebi.ac.uk 2023-03-20T13:18:08Z active site residue residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:29Z Gly(-1) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:59Z active complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP INTRO paragraph 2246 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. 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. Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. 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. 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. 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). In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. 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. 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. site SO: melaniev@ebi.ac.uk 2023-03-20T13:22:13Z substrate-binding channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:59Z active protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits site SO: melaniev@ebi.ac.uk 2023-03-20T16:02:57Z active sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:01Z Thr1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:22:47Z complex chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:23:11Z enzyme chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:14Z peptide complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T13:23:19Z N-terminal nucleophilic (Ntn) hydrolases protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:23:26Z free residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:07Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:23:35Z autocatalytic precursor processing protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:23:43Z immature site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:01Z active sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:10Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:23:59Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonding residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:14Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:24:40Z autocatalytic removal structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:28Z active-site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:24:46Z biochemical and structural analyses protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:13Z active site ptm MESH: melaniev@ebi.ac.uk 2023-03-20T11:54:48Z autocatalytic activation complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:03Z Thr residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:17Z Ser experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:25:21Z X-ray crystallography experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:25:25Z activity and inhibition assays RESULTS title_1 4066 Results RESULTS title_2 4074 Inactivation of proteasome subunits by T1A mutations complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome protein PR: melaniev@ebi.ac.uk 2023-03-20T13:25:52Z subunits mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:26:07Z T1A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:26:13Z mutations RESULTS paragraph 4127 Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. 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. 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). These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. 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). 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). complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z Proteasome taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:13Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:28:44Z mutation to residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:19Z active sites taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z Yeast mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:29:45Z catalytic protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:29:56Z disabled protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:05Z carry remnants of structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:42Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:26:07Z T1A protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:31Z deleted but expressed separately protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:46Z crystallographic analysis mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:50Z mutation site SO: melaniev@ebi.ac.uk 2023-03-20T13:31:01Z catalytic active site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:31:13Z trans-expressed protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:31:19Z not bound protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:31:22Z substrate-binding channel RESULTS paragraph 5343 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. 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). 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. 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). mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:23Z Sequencing of the plasmids taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:27Z site-directed mutagenesis mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:38Z K81R protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:31Z This single amino-acid exchange site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:23Z interface protein PR: melaniev@ebi.ac.uk 2023-03-20T13:33:46Z α4 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:33:53Z β4 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A RESULTS title_2 6407 Propeptide conformation and triggering of autolysis structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z Propeptide ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:49Z autolysis RESULTS paragraph 6459 In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. 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). 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.). 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). In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). 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. 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. 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). Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. 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. Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. 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. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:39:00Z autocatalytically cleaved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:22Z mature protein PR: melaniev@ebi.ac.uk 2023-03-20T13:39:26Z β-subunit domains protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:42Z β1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:13Z (-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:39:36Z S1 specificity pocket site SO: melaniev@ebi.ac.uk 2023-03-20T13:39:40Z substrate-binding channel residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:44Z 45 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:10Z prosegment structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:16Z antiparallel β-sheet site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:41:13Z Gly(-1) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:21Z γ-turn conformation bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:40:35Z Leu(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:40:44Z Thr1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:50Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:40:54Z crystallographically evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:40:57Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:41:04Z β1-T1A-β2-T1A protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:41:10Z Gly(-1) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:41:19Z sharp turn ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:41:36Z prosegment cleavage structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:41:47Z γ-turn conformation bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:41:55Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:41:59Z Thr(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:10Z Leu(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:17Z Gly45 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:20Z Thr(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:31Z Gly(-1) bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonding residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:31Z Thr1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:36Z Gly(-1) residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:42:40Z histidine residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:45Z (-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:49Z S2 site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:42:55Z antiparallel β-sheet residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:42:59Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:43:02Z Thr1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:38Z K81R site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:43:42Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:43:45Z Arg81 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:10Z Met45 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:43:57Z His(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:44:04Z S1 site ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:44:22Z autolysis RESULTS paragraph 8870 Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). 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). 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). Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. 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. residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:46:37Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:46:40Z autolysis taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:46:46Z His residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:46:49Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:46:53Z histidine site SO: melaniev@ebi.ac.uk 2023-03-20T13:47:01Z catalytic triads protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:09Z serine proteases residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:47:13Z His(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:16Z exchanging it for residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:47:19Z Asn residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:47:22Z Lys residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:47:25Z Phe residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:35Z H(-2)N mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:41Z H(-2)F mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:35Z H(-2)N mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:41Z H(-2)F protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:47:50Z yCPs experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:47:55Z Structural analyses structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:47:50Z yCPs evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:03Z 2FO–FC electron densities residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:06Z Gly(-1) residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:48:10Z Phe residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:13Z Lys(-2) residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:18Z Asn(-2) protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:25Z processed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:31Z unprocessed protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T13:48:36Z autolysis residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:40Z (-2) RESULTS paragraph 10064 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. 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. As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). 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). 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). 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. 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). This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 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. residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:52:19Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:22Z creating mutants that combine mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:26:07Z T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:33:38Z K81R experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:26Z mutation(s) mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:32Z H(-2)L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:40Z H(-2)T mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:48Z H(-2)A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:52:51Z substitutions residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:52:55Z Leu(-2) taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:01Z Thr(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:03Z Ala(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:59Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:10Z Met45 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:06:35Z yeasts evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:26Z crystal structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:40Z β5-H(-2)L-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:52Z β5-H(-2)T-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:54:05Z β5-H(-2)A-T1A-K81R mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:54:05Z β5-H(-2)A-T1A-K81R residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:54:15Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:54:17Z Ala(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:54:23Z Ala(-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T13:54:38Z substrate-binding channel structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:44Z prosegments mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:40Z β5-H(-2)L-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:52Z β5-H(-2)T-T1A evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:54:57Z 2FO–FC electron-density maps structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:05Z His(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:16Z Leu(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:23Z Thr(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:39:36Z S1 specificity pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:10Z Met45 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:38Z His(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T13:44:04Z S1 site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:45Z Gly(-1) protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:52Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:05Z Thr1 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:09Z (-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:56:12Z antiparallel β-sheet ptm MESH: melaniev@ebi.ac.uk 2023-03-20T16:01:52Z autolysis structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide RESULTS paragraph 12011 Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. 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. 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). Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. 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). These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:58:43Z chimeric complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:58:40Z replaced by protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:58:56Z counterpart evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:04Z 2FO–FC electron density protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:10Z Thr(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:13Z Leu(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:16Z not conserved experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:59:20Z created mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:59:23Z β2-T(-2)V complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:26Z crystal structures residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:33Z Thr(-2) bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:31Z Gly(-1) mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:52Z β5-H(-2)T-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T13:59:50Z exchange residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:59:53Z Thr(-2) residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:59:56Z Val protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:04Z 2FO–FC electron-density map protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:10Z Val(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:44:04Z S1 site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:15Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 site SO: melaniev@ebi.ac.uk 2023-03-20T14:00:21Z active-site residues residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:19Z Gly(-1) complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome RESULTS title_2 13352 The active site of the proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome RESULTS paragraph 13386 Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. 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. Owing to its likely protonation at neutral pH, however, it was assumed not to act as the general base. 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. 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. 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). The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). 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). 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. This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). 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). The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). 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). 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. 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). This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. 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. Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). 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). site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:11Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:07:50Z Thr1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:23:43Z immature complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:07Z particle residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:28Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:32Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:35Z Lys33 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonding residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:41Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:44Z catalytically active protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits site SO: melaniev@ebi.ac.uk 2023-03-20T14:08:55Z catalytic tetrad residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:08:58Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:09:01Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:09:04Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:09:07Z Asp17 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:09:15Z X-ray structures site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:09:22Z Mutation protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:06:59Z structural and biochemical analyses ptm MESH: melaniev@ebi.ac.uk 2023-03-20T16:01:56Z propeptide cleavage mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:06:15Z β5-K33A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:58Z yeast mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:34Z L(-49)S protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:40Z β5-K33A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:44Z Structural comparison mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:47Z β5-L(-49)S-K33A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:28Z active sites experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:52Z mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:28Z active-site protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:11:58Z β5-K33A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:12:02Z expressed separately chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:12:27Z β5-K33A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:12:30Z in complex with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:32Z active sites ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:12:42Z acetylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:12:53Z β5-K33A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:49:49Z apo evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:12:58Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:13:01Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:13:04Z Thr1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:13:07Z β5-K33A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:13:10Z without inhibitor residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:13:15Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z coordinates chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:13:47Z water chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:14:01Z Arg19 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:14:05Z Asp17 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:14:08Z Lys33 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:14:11Z structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:14:15Z autolysis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:14:18Z Conservative substitution residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:14:21Z Arg ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:14:24Z autolysis protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:28Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:47:50Z yCPs residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:14:35Z Arg33 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:37Z Asp17 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site RESULTS paragraph 16924 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). 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. 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). In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. 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. 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). 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. 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. In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. 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). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:20:48Z conservative mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:38Z Asp17 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:20:51Z Asn protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:20:57Z L(-49)S protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:21:00Z β5-D17N protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:21:04Z crystallographic analysis protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:21:33Z partially processed complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:21:47Z expression protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:21:51Z isolation experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:21:54Z crystallization mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:01Z D17N protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:05Z β5-D17N chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:10Z N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:17Z Suc-LLVY-AMC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:21Z carboxybenzyl-Gly-Gly-Leu-para-nitroanilide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:27Z Z-GGL-pNA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:38Z Asp17 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:22Z mature complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:33Z β5-D17N chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:22:36Z co-crystal structure chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:35:36Z α′, β′ epoxyketone chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:44Z β5-D17N chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:22:47Z in complex with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:53Z Autolysis mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:22:56Z β5-D17N residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:19Z Asn17 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:23:02Z E17A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-20T16:06:41Z β-subunit taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:23:10Z archaeon species MESH: melaniev@ebi.ac.uk 2023-03-20T14:23:15Z Thermoplasma acidophilum ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:23:18Z autolysis evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:23:22Z X-ray data mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:23:25Z β5-D17N protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:23:27Z expressed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans RESULTS paragraph 19094 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). 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. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:24:38Z CPs site SO: melaniev@ebi.ac.uk 2023-03-20T12:00:53Z catalytic triad residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:24:46Z Asp residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:24:52Z Glu17 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:24:56Z autocatalytic precursor processing chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:22Z mature protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:25:17Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:25:02Z Thr1 RESULTS paragraph 19489 To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. 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). 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). Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. 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. These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:28Z active-site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:28:38Z exchanged the conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:47Z Asp166 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:28:43Z Asn taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:28:46Z Asp166 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonded residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:28:49Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:28:52Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:28:55Z Ser169 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:28:57Z β5-D166N chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:33:19Z cis taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:12Z X-ray data mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:29:15Z β5-D166N protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:18Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:24Z Asn166 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:27Z bound to residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:29Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:32Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:29:35Z precursor processing bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:29:40Z Ser169 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:29:43Z Soaking mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:29:46Z β5-D166N experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:29:49Z crystals chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:29:55Z MG132 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:30:07Z carfilzomib complex evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:09Z structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:12Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:14Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:31:06Z Ser129 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:25Z MG132-bound state residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:28Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:30Z unmodified residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:31:06Z Ser129 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonded chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:25Z Asn166 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:30:40Z Asn residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:47Z Asp166 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:28:50Z Ala ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:30:55Z autolysis residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:47Z Asp166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:31:06Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:31:00Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:30:58Z autolysis RESULTS title_2 21052 Substitution of the active-site Thr1 by Cys experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:31:37Z Substitution site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:28Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys RESULTS paragraph 21096 Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. 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. In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). 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). 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. 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. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:33:59Z Mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T11:54:41Z 20S proteasome taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:23:10Z archaeon species MESH: melaniev@ebi.ac.uk 2023-03-20T14:33:51Z T. acidophilum taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:33:56Z mutation structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:34:09Z X-ray structure protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:34:34Z cleaved structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:34Z propeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:34:38Z still bound site SO: melaniev@ebi.ac.uk 2023-03-20T14:34:41Z substrate-binding channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:34:44Z His(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T14:34:47Z S2 pocket mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:34:57Z T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T14:35:00Z antiparallel β-sheet chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:29:55Z MG132 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:35:05Z T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:35:12Z autolysis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:35:16Z crystallization protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:00Z β5 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide RESULTS paragraph 22121 Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). 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. 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). This presumably results from the larger radius of the sulfur atom compared with oxygen. Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:39:29Z propeptide hydrolysis mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:40:08Z β5-T1C site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:12Z catalytically inactive experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:40:15Z soaking crystals complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:36Z carfilzomib protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:37Z active sites mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:40:26Z β5-T1C site SO: melaniev@ebi.ac.uk 2023-03-20T14:40:29Z proteolytic centres protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:31Z unmodified protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:34Z cleaved structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:41Z structural data residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:44Z Cys1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:24:38Z CPs residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:40:55Z Cys1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:04Z 2FO–FC electron-density map mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:40:58Z T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:31Z Lys33 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:41:11Z disordered residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:35Z Lys33 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds RESULTS title_2 23124 The benefit of Thr over Ser as the active-site nucleophile residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:04Z Thr residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:17Z Ser RESULTS paragraph 23183 All proteasomes strictly employ threonine as the active-site residue instead of serine. 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). 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). 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). 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). complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:12Z proteasomes protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:04:29Z strictly employ residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:45:18Z threonine site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:40Z active-site residue residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:45:23Z serine mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:45:27Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:45:30Z Activity assays protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:17Z Suc-LLVY-AMC mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:45:37Z T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:16Z proteasomes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:27Z Z-GGL-pNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z Crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:45:42Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:45:45Z precursor processing complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:46:11Z ligand-complex evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:46:17Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:46:22Z Ser1 RESULTS paragraph 24059 However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. This renders it unavailable for direct nucleophilic attack onto incoming substrates and first requires its reorientation, which is expected to delay substrate turnover. 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. 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). Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. 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. Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. 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.). protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:49:49Z apo evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:34:21Z crystal structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:49:55Z Ser1 site SO: melaniev@ebi.ac.uk 2023-03-20T14:49:59Z substrate-binding channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:42Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:04Z structures residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:06Z Ser1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:16Z Ser1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonded residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:25Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 site SO: melaniev@ebi.ac.uk 2023-03-20T14:50:30Z active-site residue residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrophobic interactions residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:40Z Thr3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:38Z Ala46 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:50:47Z Ser1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T14:50:50Z T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:51:08Z inhibitor complex evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:13Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:59Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:16Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:19Z with the same inhibitors evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:23Z affinity chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib site SO: melaniev@ebi.ac.uk 2023-03-20T14:51:30Z substrate-binding channel chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:34Z mean residence time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:51:37Z Ser1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:51:43Z threonine complex_assembly GO: melaniev@ebi.ac.uk 2023-03-22T10:36:40Z proteasomes protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T14:52:12Z proteasome-like proteases protein PR: melaniev@ebi.ac.uk 2023-03-20T14:52:15Z HslV DISCUSS title_1 25916 Discussion DISCUSS paragraph 25927 The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. 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. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T11:54:41Z 20S proteasome complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T14:53:53Z non-lysosomal protease taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic protein PR: melaniev@ebi.ac.uk 2023-03-20T16:06:46Z β-subunit structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:54:03Z N-acetylation structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:44Z prosegments protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:54:13Z archaeal complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:54:17Z heterologously expressed species MESH: melaniev@ebi.ac.uk 2023-03-20T14:54:25Z Escherichia coli taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:54:32Z eukaryotes protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T14:54:37Z removal of protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:10Z prosegment protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP DISCUSS paragraph 26801 Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. 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. 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. 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. 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). evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:56:42Z atomic structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:30:17Z β5-T1A protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:56:46Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:37:05Z Gly(-1) evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:56:49Z structures residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:56:52Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T14:56:58Z tight turn structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:10Z prosegment protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:57:01Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:57:05Z autolysis ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:57:09Z N-acetylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:57:13Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T14:57:17Z substrate-binding channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:57:20Z Gly(-1) residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:57:22Z (-2) DISCUSS paragraph 27707 Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. 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. 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. 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). Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:00Z CP ptm MESH: melaniev@ebi.ac.uk 2023-03-20T14:59:25Z propeptide cleavage complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T14:59:29Z CP:ligand complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T14:59:33Z active-site architecture site SO: melaniev@ebi.ac.uk 2023-03-20T12:00:54Z catalytic triad site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:01Z CP residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T14:59:41Z Asp residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:24:52Z Glu17 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:14:15Z eukaryotic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:59:54Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:54:13Z archaeal complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:59:59Z Lys33 ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:00:23Z autocatalytic removal structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:00:28Z Asp17 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:00:31Z Lys33 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:00:43Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:00:46Z Asp17 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T15:00:48Z Thr–Lys–Asp triad protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T15:00:52Z L-asparaginase residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:00:55Z Lys DISCUSS paragraph 28792 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). 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.). Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). In all proteases, collapse of the tetrahedral transition state results in selective breakage of the substrate amide bond, while the covalent interaction between the substrate and the enzyme persists. 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. 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. site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:18:47Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:18:59Z endoproteolytic cleavage residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:19:02Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:19:22Z fellutamide B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:19:25Z α-ketoamides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:19:27Z homobelactosin C chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:19:30Z salinosporamide A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:19:38Z omuralide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:19:48Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:19:51Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bridged ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:19:57Z acetylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:20:13Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:20:16Z Thr1 DISCUSS paragraph 30357 During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. 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%. The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). This renders the N terminus less suitable to stabilize substrates and to protonate the first cleavage product during catalysis, although it favours its ability to act as a nucleophile. 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. Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. 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. ptm MESH: melaniev@ebi.ac.uk 2023-03-20T16:02:02Z autolysis residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:22:46Z aspartic acid 166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:22:49Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:22:52Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:31:06Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:30:47Z Asp166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:22:56Z Thr1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:01Z mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:03Z D166A ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:06Z autolysis taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:54:13Z archaeal complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:01Z CP experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:09Z exchange mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:12Z D166N taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:01Z CP experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:15Z mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:17Z D166N residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:19Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:22Z Thr1 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:23:25Z β5-D166N protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:45Z second triad residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:29Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:32Z Asp17 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:34Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:23:37Z Asp166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 DISCUSS paragraph 31717 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. Consequently, efficient substrate turnover or covalent modification by ligands is prevented. However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. 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. residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:38Z Asp17 site SO: melaniev@ebi.ac.uk 2023-03-20T12:00:54Z catalytic triad evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:25:23Z crystallographic data protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:25:26Z proteolytically inactive mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:25:29Z β5-T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:25:35Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:05:49Z Cys1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:25:38Z cleaved ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:25:41Z autolysis protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T15:25:45Z Ntn hydrolase protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T15:25:47Z penicillin acylase experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:25:50Z substitution protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:29:45Z catalytic residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:17Z Ser residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:10Z Cys protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:25:58Z inactivates protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T15:26:01Z enzyme ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:26:03Z precursor processing DISCUSS paragraph 32443 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. Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. 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). 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. 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). 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. Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active 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. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:14:01Z CP residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:30:21Z threonine site SO: melaniev@ebi.ac.uk 2023-03-20T15:30:24Z active-site residue mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:27Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:34Z biochemically and structurally experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:37Z Activity assays mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:39Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:17Z Suc-LLVY-AMC mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:43Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T13:34:28Z yCP complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:30:47Z Structural analyses mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:30:50Z T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:30:54Z strict use of residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:04Z Thr complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:21Z proteasomes residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrophobic interactions residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:05Z Ala46 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:12Z Thr3 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:16Z proteolytically active complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:23Z proteasome taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:19Z archaea taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:25Z mammals residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:04Z Thr residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:31:32Z Ile residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:31:35Z 3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:32:02Z Ser1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T15:31:55Z enzyme protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T15:32:06Z threonine aspartase protein PR: melaniev@ebi.ac.uk 2023-03-20T15:32:09Z Taspase1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:32:12Z mutation site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:32:20Z Thr234 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:17Z Ser residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:32:23Z Thr234 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:32:26Z serine protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:51Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:02:01Z active residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:32:32Z threonine complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:24Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:32:35Z threonine complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:24Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:32:38Z conservation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:25Z proteasomes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:32:41Z bacteria taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:54:32Z eukaryotes METHODS title_1 34241 Methods METHODS title_2 34249 Yeast mutagenesis METHODS paragraph 34267 Site-directed mutagenesis was performed by standard techniques using oligonucleotides listed in Supplementary Table 2. The pre2/doa3 (β5) mutant alleles in the centromeric, TRP1- or LEU2-marked shuttle vectors YCplac22 and pRS315, respectively, were verified by sequencing and subsequently introduced into the yeast strains MHY784 (ref.) or YWH20 (ref.), which express WT PRE2 from a URA3-marked plasmid. Counter-selection against the URA3 marker with 5-fluoroorotic acid yielded strains expressing only the mutant forms of β5. METHODS paragraph 34801 The strain producing a processed β5-T1A variant and the β5 propeptide in trans is a derivative of YWH212 (ref.). It carries an additional deletion of the NAT1 gene to avoid N-acetylation of Ala1; this strain exhibits extremely slow growth rates and served for crystallographic analysis only. All strains used in this study are listed in Supplementary Table 3. METHODS title_2 35167 Purification of yeast proteasomes METHODS paragraph 35201 Yeast strains were grown in 18-l cultures at 30 °C in YPD into early stationary phase, and the yCPs were purified according to published procedures. In brief, 120 g yeast cells were solubilized in 150 ml of 50 mM KH2PO4/K2HPO4 buffer (pH 7.5) and disrupted with a French press. Cell debris were removed by centrifugation for 30 min at 21,000 r.p.m. (4 °C). The resulting supernatant was filtered and ammonium sulfate (saturated solution) was added to a final concentration of 30% (v/v). This solution was loaded on a Phenyl Sepharose 6 Fast Flow column (GE Healthcare) pre-equilibrated with 1 M ammonium sulfate in 20 mM KH2PO4/K2HPO4 (pH 7.5). CPs were eluted by applying a linear gradient from 1 to 0 M ammonium sulfate. Proteasome-containing fractions were pooled and loaded onto a hydroxyapatite column (Bio-Rad) equilibrated with 20 mM KH2PO4/K2HPO4 (pH 7.5). Elution of the CPs was achieved by increasing the phosphate buffer concentration from 20 to 500 mM. Anion-exchange chromatogaphy (Resource Q column (GE Healthcare), elution gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl (pH 7.5)) and subsequent size-exclusion chromatography (Superose 6 10/300 GL (GE Healthcare), 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) resulted in pure CPs for crystallization and activity assays. METHODS title_2 36530 Fluorescence-based activity assay METHODS paragraph 36564 ChT-L (β5) activity of CPs was monitored by fluorescence spectroscopy using the model substrate Suc-LLVY-AMC. Purified yCPs (66 nM in 100 mM Tris-HCl, pH 7.5) were incubated with 300 μM substrate for 1 h at room temperature or 37 °C. The reactions were stopped by diluting samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by proteasomal activity were measured in triplicate with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at λexc=360 nm and λem=460 nm. METHODS title_2 37088 Inhibition assays METHODS paragraph 37106 Purified yCPs were mixed with dimethylsulfoxide as a control or serial dilutions of inhibitor and incubated for 45 min at room temperature. A final concentration of yCP of 66 nM was used. After addition of the peptide substrate Suc-LLVY-AMC to a final concentration of 200 μM and incubation for 1 h at room temperature, the reaction was stopped by diluting the samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by residual proteasomal activity were measured in triplicate at λexc=360 nm and λem=460 nm. Relative fluorescence units were normalized to the dimethylsulfoxide-treated control. The calculated residual activities were plotted against the logarithm of the applied inhibitor concentration and fitted with GraphPad Prism 5. The IC50 value, the ligand concentration that leads to 50% inhibition of the enzymatic activity, was deduced from the fitted data. METHODS title_2 38002 Crystallization and structure determination METHODS paragraph 38046 Mutant yCPs were crystallized as previously described for WT 20S proteasomes. Crystals were grown at 20 °C using the hanging drop vapour diffusion method. Drops contained a 1:1 mixture of protein (40 mg ml−1) and reservoir solution (25 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES; pH 6.8) and 9–13% (v/v) 2-methyl-2,4-pentanediol (MPD)). Crystals were cryoprotected by addition of 5 μl cryobuffer (20 mM magnesium acetate, 100 mM MES, pH 6.8, and 30% (v/v) MPD). Inhibitor complex structures were obtained by incubating crystals in 5 μl cryobuffer supplemented with bortezomib or carfilzomib at a final concentration of 1.5 mM for at least 8 h. METHODS paragraph 38746 Diffraction data were collected at the beamline X06SA at the Paul Scherrer Institute, SLS, Villigen, Switzerland (λ=1.0 Å). Evaluation of reflection intensities and data reduction were performed with the programme package XDS. Molecular replacement was carried out with the coordinates of the yeast 20S proteasome (PDB entry code: 5CZ4) by rigid body refinements (REFMAC5; ref.). MAIN and COOT were used to build models. TLS (Translation/Libration/Screw) refinements finally yielded excellent Rwork and Rfree, as well as root mean squared deviation bond and angle values. The coordinates, proven to have good stereochemistry from the Ramachandran plots, were deposited in the RCSB Protein Data Bank (Supplementary Table 1). METHODS paragraph 39475 The coordinates for the yeast 20S proteasome deposited under the entry code 1RYP do not represent the WT yCP but the double-mutant β5-K33R β1-T1A. At the time of deposition (in 1997), these data were the best available on the yCP. As yCP structure determination has become routine today, and structure refinement procedures have significantly improved, we here provide coordinates for the WT yCP at 2.3 Å resolution (PDB entry code: 5CZ4). Furthermore, the structures of most mutant yCPs described in this work were determined in their apo and ligand-bound states. For mutants with proteolytically inactive β5 subunits, the best crystallographic data obtained are given. For ligands or propeptide segments that were only partially defined in the 2FO–FC electron-density map the occupancy was reduced (for details see Supplementary Table 1). 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Crystallogr. ref 69 2013 44262 MAIN software for density averaging, model building, structure refinement and validation 486 501 surname:Emsley;given-names:P. surname:Lohkamp;given-names:B. surname:Scott;given-names:W. G. surname:Cowtan;given-names:K. 20383002 REF Acta Crystallogr. D Biol. Crystallogr. ref 66 2010 44351 Features and development of Coot SUPPL footnote 44384 Author contributions E.M.H., W.H., X.L., C.S.A. and M.H. created yeast mutants; E.M.H. and W.H. performed activity and growth assays; E.M.H. and M.G. collected and analysed X-ray data; E.M.H., M.H. and M.G. wrote the manuscript. ncomms10900-f1.jpg f1 FIG fig_title_caption 44613 Conformation of proteasomal propeptides. structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides ncomms10900-f1.jpg f1 FIG fig_caption 44654 (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. The major determinant of the S1 specificity pocket, residue 45, is depicted. 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). The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). (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. Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). The major determinant of the S1 specificity pocket, residue 45, is depicted. (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:40:12Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:04:34Z matured protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:22Z (-5) to (-1) mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T13:39:36Z S1 specificity pocket residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:29Z 45 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:33Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:38Z Ala1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:40Z G(-1) protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:48:25Z processed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:29Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:50Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:40:52Z Gly(-1) experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:40:55Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:01Z Ala1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:03Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:26Z Thr(-2) bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonded residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:31Z Gly(-1) site SO: melaniev@ebi.ac.uk 2023-03-20T13:39:36Z S1 specificity pocket residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:17Z 45 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:41:20Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:25Z (-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:43Z β1 protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:51Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:45Z prosegments site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:30Z His(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide site SO: melaniev@ebi.ac.uk 2023-03-20T15:41:34Z S2 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:37Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:41:40Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:55:26Z Thr(-2) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:31Z Gly(-1) ncomms10900-f2.jpg f2 FIG fig_title_caption 46013 Mutations of residue (-2) and their influence on propeptide conformation and autolysis. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:42:07Z Mutations residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:42:09Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:42:11Z autolysis ncomms10900-f2.jpg f2 FIG fig_caption 46101 (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket. (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. While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. 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). (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. 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). For all panels stereo views are provided in Supplementary Fig. 4g–j. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:44:34Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:40Z β5-H(-2)L-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:44:40Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:45Z prosegments site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:44:43Z Structural superposition protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:18Z propeptides mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:40Z β5-H(-2)L-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:53Z β5-H(-2)T-T1A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:06:20Z β5-(H-2)A-T1A-K81R mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:30Z proteasomes residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:44:51Z (-2) to (-4) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:44:53Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:44:56Z Ala1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:00Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:45:02Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:53:53Z β5-H(-2)T-T1A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:10Z (-2) structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:17:45Z prosegments site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:18Z Thr(-2) protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:52Z β2 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bridge residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:18:31Z Gly(-1) experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:45:27Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:04:38Z matured protein PR: melaniev@ebi.ac.uk 2023-03-20T13:16:52Z β2 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:16Z β2-T1A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:06:24Z β2-T(-2)V protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:33Z Val(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:40Z Gly(-1) residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:43Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:45:45Z Gly(-1) ncomms10900-f3.jpg f3 FIG fig_title_caption 47199 Architecture and proposed reaction mechanism of the proteasomal active site. site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site ncomms10900-f3.jpg f3 FIG fig_caption 47276 (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. 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. (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. 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. Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. 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. Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. 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. Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). Substrate processing starts with nucleophilic attack of the carbonyl carbon atom of the scissile peptide bond. 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. The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. site SO: melaniev@ebi.ac.uk 2023-06-15T10:46:14Z Hydrogen-bonding network protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:39:22Z mature protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:06Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen-bonded residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:13Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:18Z Asp17 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:23Z Ser129 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:27Z 168 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:29Z Ser169 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:32Z Asp166 site SO: melaniev@ebi.ac.uk 2023-03-20T15:49:36Z active-site residues bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonding protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:49:41Z strictly conserved site SO: melaniev@ebi.ac.uk 2023-03-20T15:49:44Z proteolytic centre experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:49:47Z superposition protein PR: melaniev@ebi.ac.uk 2023-03-20T13:15:27Z β subunits experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:49:50Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:49:55Z β5-K33A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:30:26Z pp protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:30:39Z trans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:38:25Z Lys33 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:19Z Lys33 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:05Z Arg19 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:07Z Asp17 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:10Z Thr1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 site SO: melaniev@ebi.ac.uk 2023-03-20T15:50:15Z active-site residues ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:50:18Z autocatalytic precursor processing complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:25Z proteasome site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide ptm MESH: melaniev@ebi.ac.uk 2023-03-20T15:50:28Z Autolysis residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:31Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:33Z Lys33 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:36Z strictly conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:39Z Gly47 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:43Z Asp166 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:45Z Ser129 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:47Z Thr1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:50:52Z Thr1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T13:23:07Z water ncomms10900-f4.jpg f4 FIG fig_title_caption 49537 The proteasome favours threonine as the active-site nucleophile. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:25Z proteasome residue_name SO: melaniev@ebi.ac.uk 2023-03-20T15:51:29Z threonine ncomms10900-f4.jpg f4 FIG fig_caption 49602 (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. (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. Relative fluorescence units were measured in triplicate after 1 h of incubation at room temperature and are given as mean values. S.d.'s are indicated by error bars. (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. The prosegment is cleaved but still bound in the substrate-binding channel. Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the 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.). The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. (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. The SH group is rotated by 74° compared with the OH group. (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. The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. Ser1 lacks this stabilization and is therefore rotated by 60°. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:54:38Z Growth tests by serial dilution protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:28:59Z yeast site SO: melaniev@ebi.ac.uk 2023-03-20T11:59:29Z active-site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:04:43Z mutants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:34Z proteasomes protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:01Z β5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:17Z Suc-LLVY-AMC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:04Z 2FO–FC electron-density map mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:54:57Z β5-T1C structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide structure_element SO: melaniev@ebi.ac.uk 2023-03-20T13:40:10Z prosegment protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:55:04Z cleaved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:55:02Z still bound site SO: melaniev@ebi.ac.uk 2023-03-20T15:55:07Z substrate-binding channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:55:11Z His(-2) site SO: melaniev@ebi.ac.uk 2023-03-20T13:42:05Z S1 pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:53:10Z Met45 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:18Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:34:12Z β5-T1A-K81R mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:21Z β5-T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:02Z β5 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:25Z Structural superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:28Z β5-T1C structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:35Z propeptide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T13:29:08Z β1-T1A site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:14Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:02Z β5 site SO: melaniev@ebi.ac.uk 2023-03-20T13:12:15Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:55:36Z in complex with complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T12:01:25Z proteasome chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:29:55Z MG132 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T15:55:41Z inhibitor structure_element SO: melaniev@ebi.ac.uk 2023-03-20T11:57:19Z propeptides site SO: melaniev@ebi.ac.uk 2023-03-20T15:55:45Z substrate-binding channel experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:48Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:02Z β5 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:55:50Z β5-T1C protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:50Z active sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:55:55Z Cys1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:04:48Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:02Z β5 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:06:30Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant site SO: melaniev@ebi.ac.uk 2023-03-20T16:03:54Z active sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:02Z Ser1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T14:00:04Z 2FO–FC electron-density map residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:09Z Ser1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:23:04Z hydrophobic interactions residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:18Z Ala46 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:21Z Thr3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:24Z Ser1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:56:44Z lacks ncomms10900-f5.jpg f5 FIG fig_title_caption 51469 Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:57:14Z β5-T1S complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:39Z proteasomes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib ncomms10900-f5.jpg f5 FIG fig_caption 51550 Inhibition assays (left panel). 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. IC50 values were determined in triplicate; s.d.'s are indicated by error bars. Note that IC50 values depend on time and enzyme concentration. Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). 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. 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. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:58:48Z Inhibition assays taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:29:00Z yeast complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:43Z proteasomes protein PR: melaniev@ebi.ac.uk 2023-03-20T13:17:02Z β5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:22:17Z Suc-LLVY-AMC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:58:51Z IC50 values evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:58:55Z IC50 values complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:47Z Proteasomes evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:58:57Z Structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T15:59:00Z β5-T1S protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:01:52Z mutant complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T15:59:04Z complex with both ligands residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:59:08Z Ser1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:54:57Z 2FO–FC electron-density maps residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:59:11Z Ser1 site SO: melaniev@ebi.ac.uk 2023-03-20T15:59:21Z P1 site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:59:18Z Leu1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T13:56:00Z WT complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:02:52Z proteasome:inhibitor complex evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T15:59:26Z structures residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:59:33Z superimposed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T15:59:35Z mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T12:00:30Z Thr1 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T13:25:17Z Ser chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:40:22Z bortezomib chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T14:12:37Z carfilzomib t1.xml t1 TABLE table_title_caption 52522 Growth phenotypes and status of autolysis and catalysis of mutants. t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><bold>Mutant</bold></th><th align="center" valign="top" charoff="50"><bold>Viability</bold></th><th align="center" valign="top" charoff="50"><bold>Temperature sensitivity</bold></th><th align="center" valign="top" charoff="50"><bold>Autolysis state of the mutant subunit</bold><xref ref-type="fn" rid="t1-fn2">*</xref></th><th align="center" valign="top" charoff="50"><bold>Activity of the mutant subunit</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β1-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β2-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β1-T1A β2-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)T-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)L-T1A</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A, pp <italic>trans</italic>, <italic>nat1</italic>Δ</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A-K81R</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A-T1A-K81R</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)T-T1A-K81R</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)L-T1A-K81R</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)K</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)F</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)N</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5pp-β1 (ref. 18)</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β2-T(-2)V</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-L(-49S)-K33A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-K33A, pp <italic>trans</italic><xref ref-type="bibr" rid="b13">13</xref></td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-F(-45)S-K33R (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D17N</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn3">†</xref></td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn3">†</xref></td></tr><tr><td align="left" valign="top" charoff="50">β5-L(-49)S-D17N</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D17N, pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D166N</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D166N, pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1S</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1C</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td></tr></tbody></table> 52591 Mutant Viability Temperature sensitivity Autolysis state of the mutant subunit* Activity of the mutant subunit WT ++++ + + +++ β1-T1A (ref.) ++++ + − − β2-T1A (ref.) +++ ++ − − β1-T1A β2-T1A (ref.) +++ ++ − − β5-T1A +/− ++++ − − β5-H(-2)A-T1A +/− ND − − β5-H(-2)T-T1A +/− ND − − β5-H(-2)L-T1A ++ ND − − β5-T1A, pp trans, nat1Δ +/− ++++ pp trans − β5-T1A-K81R + ++++ − − β5-H(-2)A-T1A-K81R +/− ND − − β5-H(-2)T-T1A-K81R +/− ND − − β5-H(-2)L-T1A-K81R ++ ND − − β5-H(-2)A ++++ ++ + +++ β5-H(-2)K ++++ ++ + +++ β5-H(-2)F ++++ +++ + ++ β5-H(-2)N ++++ +++ + ++ β5pp-β1 (ref. 18) ++++ + +/− +/− β2-T(-2)V ++++ + − − β5-L(-49S)-K33A (ref.) + ++++ − − β5-K33A, pp trans + ++++ pp trans +/− β5-F(-45)S-K33R (ref.) ++ ++++ + − β5-D17N +/− ++++ ND† ND† β5-L(-49)S-D17N + ++++ +/− +/− β5-D17N, pp trans + ++++ pp trans +/− β5-D166N ++ ++++ + +/− β5-D166N, pp trans +++ ++++ pp trans +/− β5-T1S +++ ++++ + ++ β5-T1C ++ ++++ + − t1.xml t1 TABLE table_footnote 53689 ND, not determined. t1.xml t1 TABLE table_footnote 53709 *The autolysis state was assessed by purification and crystallization of the mutant proteasomes. t1.xml t1 TABLE table_footnote 53806 †Purification of this mutant proteasome was not possible.