Abstract
Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the β5 and β2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.
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Acknowledgements
We thank J. Roelofs (University of Kansas Medical Center) for the Pba1/2 antibody; H. Schnell for early contributions to the project; and B. Schulman, F. Adolf, J. Du, E. Goodall, J. W. Harper and D. Finley for helpful discussions. This work was supported by National Institutes of Health grants R01-GM144367 and R01-GM135337 (to J.H.), and R35-GM145249 (to L.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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B.V., J.H., L.A., E.F.P., M.B., A.R., T.R. and D.F. performed the biochemical aspects of the work. R.M.W. and S.R. performed cryo-EM sample preparation, data collection, data processing, model building and refinement. R.M.W., S.R. and A.R. performed the data analysis. F.J. and L.H. performed cross-linking mass spectrometry. A.R. and J.H. prepared the figures. J.H. wrote the paper with assistance from B.V., R.M.W. and S.R., and input from all authors.
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Extended data
Extended Data Fig. 1 Conservation of midline stabilizing aspartates and arginines.
a, Comparison of the C-termini for all 7 yeast β-subunits. Only β3 and β6 end in aspartate. b, Evolutionary conservation of the β3/β5 midline salt bridge residues, β3-D205 and β5-R94. β5′s nearby catalytic triad residue, D92, is shown for reference. c, Evolutionary conservation of the β2/β6 midline salt bridge residues, β6-D241 and β2-R94. β2′s nearby catalytic triad residue, D46, is shown for reference.
Extended Data Fig. 2 Cryo-EM classification of CP species.
Shown is the processing scheme for classification and refinement of proteasome species. ‘Junk” classes are colored grey while identifiable species are colored by species. The asterisk denotes cryoDRGN classification - displayed classes show representative maps generated via k-means clustering and are not necessarily correlated to the particle selection carried out by clustering of the latent embeddings. All other 3D classification steps were carried out in cryoSPARC.
Extended Data Fig. 3 Cryo-EM data analysis for CP species.
a, Representative micrograph of proteasome particles embedded in vitreous ice. Scale bar, 500 Å. A total of 375,915 individual particles were analyzed. b, Selected 2D class averages of 20S, preholoproteasome, and Blm10-CP particles. c, Proteasome reconstructions were filtered and colored by local resolution (left panels), gold-standard Fourier shell correlation (FSC) curves from cryoSPARC (center panels), and viewing direction distribution plots (right panels). Resolution was determined at FSC = 0.143.
Extended Data Fig. 4 Comparison of Ump1 before and after CP fusion.
a, Molecular model of Ump1 from pre-15S (PDB: 7LS6) and the β3-D205Δ preholoproteasome. The first and last resolved residues are shown. b, Overlay of the two models, showing no significant conformational changes.
Extended Data Fig. 5 Detailed analysis of the β5-propeptide.
a, Comparison of β5 between mature wild-type 20S (PDB: 5CZ4) and the β3-D205Δ preholoproteasome. The mature portions of β5 largely overlap, although there is a slight rotation of β5 towards the CP midline in the mature 20S, which may reflect final tightening of the CP upon completion of assembly. The first and last resolved residues are indicated. b, Overlay of the two structures from panel A, highlighting the differences between them. Arrows designate portions of β5 that are unresolved in preholoproteasome. c, Overlay of the molecular model of the β5-propeptide onto its primary map density, confirming the validity of the model. Boxed panels show close-up views of selected regions and arrows indicate their position in the overall propeptide.
Extended Data Fig. 6 Validation of the β5 Propeptide Structure by Crosslinking Mass Spectrometry.
Crosslinking mass spectrometry was performed on β3-D205Δ proteasomes, which identified two crosslinks involving the β5-propeptide: β5-D61/D62 with β5-D193, and β5-E27 with α5-E131. A third crosslink, β5-K16 with α6-K115, was detected in a prior study13. All three crosslinks are within the crosslinkable distance for the respective Lys-Lys and Asp/Glu-Asp/Glu crosslinkers, and strongly support the modeled structure of the β5-propeptide. The orange dashed lines indicate the crosslinked residues. The other colored dashed lines indicate unresolved residues.
Extended Data Fig. 7 Graphical Representation of the Crosslinking Mass Spectrometry Data for the Propeptides, Pba1/2, and Ump1.
Intersubunit (green lines) and intrasubunit (blue lines) crosslinks are shown for the β-subunit propeptides (panel A), Pba1/2 (panel B), and Ump1 (panel C). Shaded areas indicate the propeptides.
Extended Data Fig. 8 Analysis of the β1 catalytic triad.
Arg38 in β1 shows a similar orientation to the corresponding arginine residues in β5 and β2, and is hydrogen bonded (dashed lines) to the triad aspartate and lysine residues, suggesting that it may play an important role in β1 activation. However, unlike the situation in β5 and β2, β1′s Arg38 does not form a salt bridge or even contact its midline partner, β7, suggesting that additional mechanisms may account for β1 activation.
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Velez, B., Walsh, R.M., Rawson, S. et al. Mechanism of autocatalytic activation during proteasome assembly. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01262-1
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DOI: https://doi.org/10.1038/s41594-024-01262-1
