Mechanism of autocatalytic activation during proteasome assembly

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. Access through your

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CHAPERONE-MEDIATED MULTISTEP ASSEMBLY OF THE HUMAN 20S PROTEASOME Article Open access 10 April 2024 STRUCTURE OF THE PREHOLOPROTEASOME REVEALS LATE STEPS IN PROTEASOME CORE PARTICLE

BIOGENESIS Article 31 August 2023 STRUCTURES OF CHAPERONE-ASSOCIATED ASSEMBLY INTERMEDIATES REVEAL COORDINATED MECHANISMS OF PROTEASOME BIOGENESIS Article 12 April 2021 DATA AVAILABILITY

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Scholar  Download references 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. AUTHOR INFORMATION Author

notes * These authors contributed equally: Benjamin Velez, Richard M. Walsh Jr, Shaun Rawson. AUTHORS AND AFFILIATIONS * Department of Pathology, Harvard Medical School and Brigham and

Women’s Hospital, Boston, MA, USA Benjamin Velez, Aida Razi, Lea Adams, Erignacio Fermin Perez, Marie Blickling, Tamayanthi Rajakumar, Darlene Fung & John Hanna * Harvard Cryo-Electron

Microscopy Center for Structural Biology, Harvard Medical School, Boston, MA, USA Richard M. Walsh Jr. & Shaun Rawson * Department of Biological Chemistry and Molecular Pharmacology,

Blavatnik Institute, Harvard Medical School, Boston, MA, USA Richard M. Walsh Jr. & Shaun Rawson * Department of Physiology and Biophysics, University of California-Irvine, Irvine, CA,

USA Fenglong Jiao & Lan Huang Authors * Benjamin Velez View author publications You can also search for this author inPubMed Google Scholar * Richard M. Walsh Jr. View author

publications You can also search for this author inPubMed Google Scholar * Shaun Rawson View author publications You can also search for this author inPubMed Google Scholar * Aida Razi View

author publications You can also search for this author inPubMed Google Scholar * Lea Adams View author publications You can also search for this author inPubMed Google Scholar * Erignacio

Fermin Perez View author publications You can also search for this author inPubMed Google Scholar * Fenglong Jiao View author publications You can also search for this author inPubMed Google

Scholar * Marie Blickling View author publications You can also search for this author inPubMed Google Scholar * Tamayanthi Rajakumar View author publications You can also search for this

author inPubMed Google Scholar * Darlene Fung View author publications You can also search for this author inPubMed Google Scholar * Lan Huang View author publications You can also search

for this author inPubMed Google Scholar * John Hanna View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to John Hanna. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER

REVIEW INFORMATION _Nature Structural & Molecular Biology_ thanks Youdong Mao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Dimitris

Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. ADDITIONAL INFORMATION PUBLISHER’S NOTE

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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. SUPPLEMENTARY INFORMATION REPORTING

SUMMARY SOURCE DATA SOURCE DATA FIG. 3G Unprocessed gel and blots. SOURCE DATA FIG. 4B,E Unprocessed gel and blots. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or

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this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Velez, B., Walsh, R.M., Rawson, S.

_et al._ Mechanism of autocatalytic activation during proteasome assembly. _Nat Struct Mol Biol_ 31, 1167–1175 (2024). https://doi.org/10.1038/s41594-024-01262-1 Download citation *

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