Abstract
Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90’s function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients.
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Data availability
The atomic coordinates for Hsp90M–Δ131Δa, Hsp90M–Δ131Δb and Hsp90–Δ131Δ were deposited to the PDB with accession codes 8K2S, 8K2R and 8K2T, respectively. The NMR assignments for Δ131Δ, FtsZC, Hsp90N, Hsp90C, Hsp90M–Δ131Δa, Hsp90M–Δ131Δb and Hsp90–Δ131Δ were deposited to the Biological Magnetic Resonance Data Bank (BMRB) under accession codes 52367, 52368, 52364, 52366, 36581, 36580 and 36582, respectively. Additionally, previously published models of Hsp90 or Hsp90–client complexes essential for building the Hsp90–Δ131Δ structure and describing the bipartite binding mode were included in this study with corresponding PDB accession codes 2IOQ, 5FWK, 7KRJ and 7KW7 and references. Other data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We gratefully acknowledge I. Chen, Y. Xia and T. Xie for insightful discussions and the Bionmr Laboratory of the Division of Life Sciences and Medicine at the University of Science and Technology of China for NMR data collection. A portion of this work was performed at the Steady High Magnetic Field Facility (High Magnetic Field Laboratory, Chinese Academy of Sciences). This study was supported by the National Natural Science Foundation of China (31770807, 31971144 and T2221005) to C.H., the National Key R&D Program of China (2023YFC3403102) to C.H. and the Anhui Provincial Natural Science Foundation (2208085MC47) to C. Wan.
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C.H. and W.X. designed the research. X.Q., S.Z., C. Wan, T.J. and W.X. prepared the protein samples and conducted biochemical experiments. X.Q., S.Z., C. Wan, L.Z. and C.H. performed the NMR experiments. C.H., X.Q., S.Z., C. Wan, P.R., J.W., C.G.K., C. Wang and W.X. analyzed the data. X.Q., P.R. and C.H. performed the structure calculations. C.H., X.Q., P.R. and C.G.K. drafted the manuscript. All authors reviewed and approved the final manuscript.
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Extended data
Extended Data Fig. 1 NMR characterization of clients.
a, 1H–15N HSQC spectrum of full-length Δ131Δ. b, Overlay of 1H–15N HSQC spectrum of full-length Δ131Δ and the fragments (Δ131ΔN and Δ131ΔC, corresponding to the sites a and b, respectively). The excellent resonance correspondence indicates that the conformational properties of isolated fragments recapitulate those of the full-length protein. c,d, Secondary structure propensity (SSP) values of Δ131Δ (c) and FtsZC (d) plotted as a function of the amino acid sequence. A SSP score at a given residue of 1 or −1 reflects a fully formed α-helical or β-structure (extended), respectively, whereas a score of, for example, 0.5 indicates that 50% of the conformers in the native-state ensemble of the protein are helical at that position. The Hsp90-binding sites are highlighted. The data from panels c and d collectively suggest that the recognition sites of Hsp90 within clients are independent of their secondary structure patterns.
Extended Data Fig. 2 Interactions between the client and various nucleotide bound states of Hsp90.
a,b, 1H–15N HSQC spectrum of Δ131Δ fragments and FtsZC in the absence and presence of AMP-PNP-bound (a) and ADP-bound (b) Hsp90. c, ITC analysis of Δ131Δ and Δ131ΔC binding to different nucleotide bound states of Hsp90. d,e, NMR titration profiles using two-state exchange models to determine the affinities of Apo form of Hsp90 and different nucleotide bound state of Hsp90 to Δ131Δ (d) and FtsZC (e). The titration profiles for selected residues are displayed on the top. These data indicate the binding affinities throughout the chaperone cycle remain largely unaltered.
Extended Data Fig. 3 Interactions of clients with Hsp90 domains.
a-c, Overlay of 1H–15N HSQC spectrum of Δ131Δ fragments and FtsZC in the absence and presence of the NTD (a), CTD (b) and MD (c) of Hsp90. These data collectively suggest that the middle ___domain alone is responsible for client recognition, with neither Hsp90N nor Hsp90C involved in binding.
Extended Data Fig. 4 Synergistic binding of the MDs of Hsp90 to clients.
a-d, Overlay of 1H–15N HSQC spectra of Δ131Δ fragments and FtsZC in the absence and presence of Hsp90NM (a), Hsp90MC (b), Hsp90MCLC (c) and the dimerized Hsp90MM (d). These data collectively demonstrate a significant binding synergy between Hsp90M and clients.
Extended Data Fig. 5 NMR characterizations and assignments of Hsp90 domains.
a, Superimposed methyl-TROSY spectra of the full-length Hsp90 with its individual domains: NTD, MD, CTD, their combinations- NM domains and MC domains. Analysis of these spectra reveals a lack of extensive contacts between the individual domains. b, Representative strips extracted from the non-uniformly sampled HNCACB experiments of the individual domains, displaying the backbone resonance assignment.
Extended Data Fig. 6 Identifications of the client-binding sites on Hsp90.
a, Overlay of the methyl-TROSY spectra of Hsp90 and the Hsp90-Δ131Δ complex. b, Overlay of 1H–15N TROSY HSQC spectra of Hsp90M and in complex with Δ131Δa, Δ131Δb and FtsZC. c, Overlay of methyl-TROSY spectra of Hsp90M in complex with Δ131Δa, Δ131Δb and FtsZC. d, Inter-molecular NOEs between Hsp90 and the Δ131Δ protein. Representative strips extracted from the 13C-edited HMQC–NOESY–HMQC NMR experiments. The NOE cross-peaks between Hsp90 and residues of Δ131Δ fragments are designated by a dashed-line red circle.
Extended Data Fig. 7 Strategy for structure determination of the Hsp90-Δ131Δ complex.
More details can be found in Methods.
Extended Data Fig. 8 Detailed contacts between Hsp90 and the Δ131Δ protein.
Inter-molecular contacts between Hsp90 and site a (a) and site b (b) generated by Ligplot.
Extended Data Fig. 9 Interactions between the clients and different Hsp90s.
a, 1H–15N HSQC spectrum of Δ131Δ fragments and FtsZC in the absence and presence of yeast Hsp90 (Hsc82). b, 1H–15N HSQC spectrum of Δ131Δ fragments and FtsZC in the absence and presence of human Hsp90 (Hsp90β). The recognition sites in Δ131Δ fragments and FtsZC are indicated. The data reveal significant overlap in the regions of the client proteins bound by different Hsp90 species, suggesting a conserved binding mechanism.
Extended Data Fig. 10 A simplified scheme illustrating the functional cycles of client chaperoning by Hsp90.
Initially, Hsp90 captures the client in an unfolded conformation in its open state, with two stretches engaged simultaneously by one hydrophobic groove on each protomer. ATP binding, assisted by client engagement and/or cochaperone associations, shifts the conformation equilibrium of Hsp90 towards clamp closure. ATP hydrolysis triggers a significant conformational change in Hsp90, transitioning it to its ADP state, and subsequent ADP release leads to the complex returning to its nucleotide free state.
Supplementary information
Supplementary Information
Supplementary Figs. 1–4.
Supplementary Data 1
Primer sequences used in this study.
Source data
Source Data Fig. 1
Source data for NMR titrations in Fig. 1f,g.
Source Data Fig. 2
Source data for NMR titrations in Fig. 2.
Source Data Fig. 6
Raw readings for the Fig. 6b,c.
Source Data Extended Data Fig. 2
Source data for NMR titrations in Extended Data Fig. 2.
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Qu, X., Zhao, S., Wan, C. et al. Structural basis for the dynamic chaperoning of disordered clients by Hsp90. Nat Struct Mol Biol 31, 1482–1491 (2024). https://doi.org/10.1038/s41594-024-01337-z
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DOI: https://doi.org/10.1038/s41594-024-01337-z
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