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Published May 10, 2023 | Published + Supplemental Material
Journal Article Open

Dodecamer assembly of a metazoan AAA⁺ chaperone couples substrate extraction to refolding


Ring-forming AAA⁺ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA⁺ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.

Additional Information

© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). We thank H. Wu (UCSF) and S. Petrovic (CalTech) for assistance with cryo-EM data analysis and structural modeling, C. Wang for construction of the luciferase expression plasmid, E. Chapman for the pTrc-GroEL_D87K plasmid, E. Deuerling for plasmids pHis₆-SUMO-Ssa1 and pUlp1-His₆, and members of the Shan and Cheng groups for discussions and comments on the manuscript. S.-o.S. was supported by National Institutes of Health (NIH) grant R35 GM136321 and a travel fund from the Burroughs Welcome Foundation. U.S.C. was supported by NIH fellowship F32GM137463. Y.C. is supported by NIH grant R35GM140847 and is an Investigator of Howard Hughes Medical Institute. Cryo-EM data were collected at UCSF cryo-EM facility, supported by NIH grants S10OD021741 and 1S10OD026881, and the Stanford-SLAC Cryo-EM Center (S²C²), supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24 GM129541). Author contributions: Conceptualization: S.-o.S. Methodology: U.S.C., Z.Y., and Y.C. Investigation: A.G., S.-o.S., A.S., A.M.L., and Z.Y. Visualization: A.M.L., A.S., and A.G. Supervision: S.-o.S., Z.Y., and U.S.C. Writing—original draft: S.-o.S., A.M.L., A.S., and A.G. Writing—review and editing: all authors. Data and materials availability: Cryo-EM maps and model coordinates are deposited in the EMDB and PDB as PDB entry ID 7US2 and EMDB entry ID EMD-26722 [high-resolution map of PARL-cleaved Skd3 (human ClpB) E455Q Nucleotide Binding Domain hexamer bound to ATPgammaS, open conformation]; EMD-26725 [3DVA derived map of PARL-cleaved Skd3 (human ClpB) E455Q Nucleotide Binding Domain hexamer bound to ATPgammaS, open conformation]; EMD-26726 [3DVA derived map of PARL-cleaved Skd3 (human ClpB) E455Q Nucleotide Binding Domain hexamer bound to ATPgammaS, closed conformation]; EMD-26728 [PARL-cleaved Skd3 (human ClpB) E455Q dodecamer bound to ATPgammaS]. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. pSG25-β-Gal (#63867) and pET-Sac-Abeta(M1-42) (#71875) can be purchased from Addgene pending a completed material transfer agreement. Competing interests: Y.C. is a member of scientific advisory board of ShuiMu BioSciences. The other authors declare that they have no competing interests.

Attached Files

Published - sciadv.adf5336.pdf

Supplemental Material - sciadv.adf5336_movie_s1.zip

Supplemental Material - sciadv.adf5336_sm.pdf


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August 22, 2023
October 25, 2023