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Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90

Nature Structural & Molecular Biology volume 24, pages 407–413 (2017)Cite this article

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Abstract

The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.

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Figure 1: Three-dimensional structure of the cytotoxic conformation of TTR.
Figure 2: The molecular chaperone Hsp90 binds to misfolded TTR.
Figure 3: The Hsp90 dimer is in an open conformation in the presence of M-TTR.
Figure 4: The metastable monomeric variant of TTR binds to the N-terminal and M domains of Hsp90.
Figure 5: TTR retains its globular structure in complex with Hsp90.
Figure 6: The intrinsically disordered protein tau and the toxic misfolded conformation of TTR share a common binding surface on Hsp90.

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Change history

  • 13 March 2017

    In the version of this article initially published online, there was an error in the y-axis label of Figure 1e. The error has been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We thank M. Mizuguchi (University of Toyama, Toyama, Japan) for the transthyretin plasmid; C.A. Dickey (University of South Florida, Tampa, Florida, USA) for the Hsp90 plasmid; and C.A. Dickey, B.A. Nordhues and S.G. Rüdiger for useful discussions. We are grateful to M. Ubbink (Leiden University, Leiden, the Netherlands) for the CLanP-7 lanthanide tag, to P. Wysoczanski for help with NMR spectroscopy experiments recorded for Hsp90's M domain, and to A. Pérez-Lara for help with ITC experiments. This work was supported by the Alexander von Humboldt Foundation (fellowship to J.H.K.), the European Commission (Marie Curie Intra-European fellowship, project number 626526 to J.O.), the Fulbright Program (scholarship to B.J.C.) and the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement 283570 to M.Z.).

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Author notes

  1. Javier Oroz and Jin Hae Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany

    Javier Oroz, Jin Hae Kim & Markus Zweckstetter

  2. Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Bliss J Chang & Markus Zweckstetter

  3. Department of Neurology, University Medical Center Göttingen, University of Göttingen, Göttingen, Germany

    Markus Zweckstetter

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  1. Javier Oroz
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Contributions

J.H.K. performed NMR spectroscopy and biochemical experiments on TTR variants, as well as structure calculations. J.O. performed NMR spectroscopy, SAXS and ITC experiments on Hsp90. B.J.C. and J.O. produced Hsp90 mutants for the assignment of isoleucine methyl groups. J.H.K., J.O. and M.Z. designed experiments. J.H.K., J.O. and M.Z. wrote the paper.

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Correspondence to Markus Zweckstetter.

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Integrated supplementary information

Supplementary Figure 1 The structure of the misfolded monomeric conformation of TTR is well defined.

(a) Comparison of 1H-15N NMR spectra of M-TTR at ambient pressure (green) and 500 bar (blue). Working at 500 bar improves the spectral quality. (b) Experimentally observed NOEs (green dotted lines) are visualized on the 3D structure of M-TTR (blue).

Supplementary Figure 2 Reproducibility in calorimetric titrations of Hsp90 with M-TTR.

From left to right and top to bottom, raw data resulting from the titration of 25x1.5 μl aliquots of 1671 μM of M-TTR into 25 μM of Hsp90 (a), 783 μM of M-TTR into 30 μM of Hsp90 (b), 520 μM of M-TTR into 30 μM of Hsp90 (c), 313 μM of M-TTR into 25,5 μM of Hsp90 (d) and 166 μM of M-TTR into 22,75 μM of Hsp90 (e) are shown. The biphasic thermodynamic behavior was present in all tested conditions.

Supplementary Figure 3 Nucleotide binding does not promote allosteric changes in the Hsp90–M-TTR complex.

(a) As observed from the P(r) distribution obtained from SAXS scattering data, ADP binding does not change the global extended conformation of the Hsp90/M-TTR complex. While Hsp90 shows Rg= 6.34 ± 0.16 nm and DMAX= 20.94 ± 0.52 nm, the Hsp90+M-TTR complex has Rg= 6.58 ± 0.44 nm and DMAX= 21.77 ± 0.71 nm. (b) Comparison of NMR signal intensities of the methyl signals of M-TTR/Hsp90 in the absence and presence of 2 mM ADP. Samples contained 0.07 mM 13C-labeled and methyl-protonated M-TTR in 50 mM MES, pH 7, 100 mM NaCl, 5 mM DTT, 1 mM MgSO4, 0.1 mM DSS, and a 2-fold (blue) or 4-fold (green) molar excess of Hsp90.

Supplementary Figure 4 Hsp90 uses several binding interfaces to bind M-TTR.

Residues in the N- and M-domain of Hsp90, which show the strongest PCSs in the presence of M-TTR, are highlighted in orange in the 3D structure. Hsp90’s N-domain is colored in light blue, M-domain in red and C-terminal domain in light green. The charged linker, which connects Hsp90’s N and M domain, is represented by a grey line.

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Supplementary Figures 1–4 and Supplementary Table 1 (PDF 868 kb)

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Oroz, J., Kim, J., Chang, B. et al. Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90. Nat Struct Mol Biol 24, 407–413 (2017). https://doi.org/10.1038/nsmb.3380

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