CaltechAUTHORS
Published July 1, 2024 | Version in press
Journal Article Open

Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly

Creators

Abstract

The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin progresses through its ATP-dependent client folding cycle are not clear. Here, we determined cryo-EM structures of a hyperstable disease-associated human mtHsp60 mutant, V72I. Client density is identified in three distinct states, revealing interactions with the mtHsp60 apical domains and C termini that coordinate client positioning in the folding chamber. We further identify an asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60–10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify distinct roles for the apical domains in coordinating client capture and progression through the chaperone cycle, supporting a conserved mechanism of group I chaperonin function.

Copyright and License

© The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Acknowledgement

We thank A. Brilot for helpful advice regarding cryo-EM data processing. This work was supported by the National Institutes of Health, grants F31GM142279 (to J.R.B.), NS059690 (to J.E.G.) and R01GM138690 (to D.R.S.).

Contributions

J.R.B. cloned mtHsp60 mutants, expressed and purified proteins, performed biochemical and cryo-EM experiments, built models, developed figures, and wrote and edited the manuscript. H.S. expressed and purified proteins. E.T. operated electron microscopes and assisted with data collection. J.E.G. and D.R.S. designed and supervised the project and wrote and edited the manuscript.

Data Availability

Cryo-EM densities have been deposited at the Electron Microscopy Data Bank (EMDB) under accession codes EMD-29813 (mtHsp60apo consensus), EMD-29814 (mtHsp60apo focus), EMD-29815 (mtHsp60ATP consensus), EMD-29816 (mtHsp60ATP focus), EMD-29817 (mtHsp60ATP–mtHsp10 consensus) and EMD-29818 (mtHsp60ATP–mtHsp10 focus). Atomic coordinates have been deposited at the Protein Data Bank (PDB) under accession codes PDB 8G7J (mtHsp60apo consensus), PDB 8G7K (mtHsp60apo focus), PDB 8G7L (mtHsp60ATP consensus), PDB 8G7M (mtHsp60ATP focus), PDB 8G7N (mtHsp60ATP–mtHsp10 consensus) and PDB 8G7O (mtHsp60ATP–mtHsp10 focus). Accession codes for additional models referenced in this study are PDB 7AZP (mtHsp60 apo), PDB 6MRC (mtHsp60ADP–mtHsp10), PDB 4KI8 (GroEL R-ADP), PDB 4AAR (GroEL Rs2), PDB 1AON (GroELADP–GroES), PDB 1SVT (GroELADP-AlFx–GroES) and PDB 1KP8 (GroELATP). Source data are provided with this paper.

Extended Data Fig. 1 Biochemical and cryo-EM analysis of apo mtHsp60V72I.

Extended Data Fig. 2 Cryo-EM densities and resolution estimation from the mtHsp60V72I datasets.

Extended Data Fig. 3 Cryo-EM analysis of ATP-bound mtHsp60V72I.

Extended Data Fig. 4 Cryo-EM analysis of ATP/mtHsp10-bound mtHsp60V72I.

Extended Data Fig. 5 Sequence alignments and structural comparisons of group I chaperonins.

Supplementary Video 1

Source Data Fig. 1

Source Data Fig. 4

Source Data Fig. 5

Source Data Extended Data Fig. 1

Source Data Extended Data Fig. 1

Conflict of Interest

The authors declare no competing interests.

Files

s41594-024-01352-0.pdf

Files (38.8 MB)

Name Size
md5:b0f0585e3cc7bf71a7a307301a2c6864
387.6 kB Preview Download
md5:94a27ff5a7ef77be43476d6cc056d0a2
633.0 kB Preview Download
md5:b6e488736c8bb0913584fe94b76ecea3
647.0 kB Preview Download
md5:5de64c2df200057542b034074fcfc9c4
643.8 kB Preview Download
md5:4c564de5c81886507454ddc0c55023b3
566.6 kB Preview Download
md5:9fb4167678b97743bc76d6eaa2a71785
20.9 MB Download
md5:37510344ea3934f5b667120e18d86cb6
149.8 kB Download
md5:3217822bebd8c37806bd243ac365e088
813.9 kB Download
md5:5642c26730a273ceea31d33caaa5e2ad
2.8 MB Download
md5:28530bfec38bdc0c20c26f564bf10649
11.5 kB Download
md5:781664fb20e6e705db4433a02b1d68c0
2.1 MB Preview Download
md5:85eab0eec229b06c178bfeb3a53be1fb
9.2 MB Preview Download

Additional details

Identifiers

ISSN
1545-9985

Funding

National Institutes of Health
NIH Postdoctoral Fellowship F31GM142279
National Institutes of Health
NS059690
National Institutes of Health
R01GM138690