Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes

Abstract

Proteases are defined by their nucleophile but require additional residues to regulate their active sites, most often arranged as catalytic triads that control the generation and resolution of acyl-enzyme intermediates. Threonine N-terminal nucleophiles represent a diverse family of proteases and transferases that possess two active site nucleophiles, the side chain hydroxyl and the free amino-terminus, and require autocatalytic cleavage of their N-terminal propeptides. Here we provide evidence that the proteasome, which mediates intracellular protein degradation and contains three different threonine protease subunits, utilizes a unique catalytic pentad mechanism. In addition to the previously defined lysine/aspartate pair which regulates threonine's side chain, a second serine/aspartate pair appears to regulate threonine's amino-terminus. The pentad is required for substrate proteolysis and assembly-coupled autocatalytic cleavage, the latter triggered by alignment of the full pentad upon fusion of two half-proteasome precursors. A similar pentad mechanism was required by the ornithine acetyltransferase Arg7, suggesting that this may be a general property of threonine N-terminal nucleophiles. Finally, we show that two patient-derived proteasome mutations compromise function of the serine/aspartate unit in yeast, suggesting that defective pentad function may underlie some human proteasomopathies.

Fig. 1. Structural analysis of β-β-ring midline interactions.

Shown are the atomic models of wild-type CP (PDB: 5CZ4) with corresponding unresolved regions from the pre-15S assembly intermediate (PDB: 7LS6) shown in various colors. Panel (a) shows the β2/β6 interface. One unresolved loop in β2 appears to regulate the canonical threonine/lysine/aspartate catalytic triad, suggesting that stabilization of this loop may be a key feature of autocatalytic activation. However, β2 contains a separate unresolved loop whose function was unknown, and which contains a highly conserved aspartate residue with a nearby conserved serine residue, both in close proximity to the active site threonine. A similar arrangement was seen for the β5/β3 pair (b).

Fig. 2. A catalytic pentad is required for autocatalytic activation and proteolysis by β2.

a Structural arrangement of the proposed pentad residues as seen in mature wild-type CP (PDB: 5CZ4). Lys62, Asp46, and Thr30 represent the canonical catalytic triad, while Ser158 and Asp195 represent additional residues proposed to complete the catalytic pentad. Dashed lines, hydrogen bonds. b Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements, in a strain where endogenous β2 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β2 locus is repressed and plasmid-derived β2 is the sole source of this protein. Plates were cultured at 30 °C for the indicated number of days. c Purified CP from wild-type and mutant strains, as analyzed using SDS-PAGE followed by Coomassie staining. Asterisk, a break-down product of α7. Note that Pba1, Pba2, and immature β5 co-migrate as a single band. d Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, Pba1/2, and immature (propeptide-bearing) β5. e Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. f Levels of transcription factor Rpn4, which senses cellular proteasome capacity, in whole cell extracts from wild-type and mutant strains, as determined by SDS-PAGE and immunoblotting. GAPDH, loading control. Similar results were obtained in 5 (b), 4 (c, d) and 2 (f) independent experiments, respectively.

Fig. 3. A catalytic pentad is required for autocatalytic activation and proteolysis by β5.

a Structural arrangement of the proposed pentad residues as seen in mature wild-type CP (PDB: 5CZ4). Lys108, Asp92, and Thr76 represent the canonical catalytic triad, while Ser206 and Asp243 represent additional residues proposed to complete the catalytic pentad. Dashed lines, hydrogen bonds. b Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β5 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β5 locus is repressed and plasmid-derived β5 is the sole source of this protein. Plates were cultured at 30 °C for 3 days. c Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, immature (propeptide-bearing) β5, and Pba1/2. d Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. e Turnover of immature β5, as determined by cycloheximide chase analysis of whole cell extracts. Analysis was by SDS-PAGE followed by immunoblotting with an antibody that specifically recognizes immature β5. GAPDH, loading control. Asterisk, non-specific band. Similar results were obtained in 4 (b) and 2 independent experiments (c and e), respectively.

Fig. 4. A catalytic pentad is required for autocatalytic activation and proteolysis by β1.

a Structural arrangement of the proposed pentad residues as seen in mature wild-type CP (PDB: 5CZ4). Lys52, Asp36, and Thr20 represent the canonical catalytic triad, while Ser148 and Asp185 represent additional residues proposed to complete the catalytic pentad. Dashed lines, hydrogen bonds. b Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β1 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β1 locus is repressed and plasmid-derived β1 is the sole source of this protein. Plates were cultured at 30 °C for 2 (no drug) and 7 days (canavanine, 1.5 μg/mL). c Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, immature (propeptide-bearing) β5, and Pba1/2. d Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. e Structures of the helix-loop-helix motif responsible for the spatial proximity of the Ser/Asp pair within the catalytic pentad (PDB: 5CZ4). Similar results were obtained in 2 independent experiments (b, c).

Fig. 5. A catalytic pentad is required for function and autocatalytic activity of the ornithine acetyltransferase Arg7.

a Structural arrangement of the proposed pentad residues as seen for Streptomyces clavuligerus ornithine acetyltransferase (PDB: 2VZK). For clarity, residue numbering is after yeast Arg7. Dashed lines represent hydrogen bonds. b Evolutionary conservation of the proposed catalytic pentad residues. c Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in the arg7Δ mutant, which is strongly (although not fully) auxotrophic for arginine. Plates were cultured at 30 °C for 2 days. d Maturation of wild-type and mutant Arg7-3xHA proteins, as determined by analysis of whole cell extracts using SDS-PAGE followed by immunoblotting with anti-HA antibody. GAPDH, loading control. e Expression of recombinant wild-type and mutant Arg7 in bacteria, as analyzed by SDS-PAGE followed by Coomassie staining. Gene expression is induced by IPTG. The dimer seen for mature Arg7 reflects autocatalytic cleavage into two comparably sized polypeptides. Similar results were obtained in 2 (b) and 3 independent experiments (d, e), respectively.

Fig. 6. Modeling of a patient-derived β5 mutation in yeast.

A Sequence alignment of human immunoproteasome β5i and yeast β5. The pentad serine residue is in cyan. B Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β5 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β5 locus is repressed and plasmid-derived β5 is the sole source of this protein. Plates were cultured at 30 °C for 2 (galactose), 1 (glucose), and 5 (canavanine 1.5 μg/mL) days. C Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, immature (propeptide-bearing) β5, and Pba1/2. D Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. E Turnover of immature β5, as determined by cycloheximide chase analysis of whole extracts. Analysis was by SDS-PAGE followed by immunoblotting with the antibody that specifically recognizes immature β5. GAPDH, loading control. Asterisk, non-specific band. Similar results were obtained in 3 (B) and 2 independent experiments (C and E), respectively.

Fig. 7. Modeling of a patient-derived β2 mutation in yeast.

A Sequence alignment of human immunoproteasome β2i and yeast β2. The pentad aspartate residue is in cyan. B Position of the corresponding G191 residue in yeast CP. An amino acid side chain at this position would be predicted to disrupt the Ser/Asp pair of the catalytic pentad. C Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β2 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β2 locus is repressed and plasmid-derived β2 is the sole source of this protein. Plates were cultured at 30 °C for 2 (galactose and glucose) and 7 (canavanine, 2 μg/mL) days. D Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with α5 antibody. E Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. Similar results were obtained in 3 (C) and 2 independent experiments (D), respectively.