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HSP40 proteins use class-specific regulation to drive HSP70 functional diversity

Nature volume 587, pages 489–494 (2020)Cite this article

Abstract

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation1,2,3. Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding3,4. In humans, JDPs constitute a large and diverse family with more than 40 different members2, which vary in their substrate selectivity and in the nature and number of their client-binding domains5. Here we show that JDPs can also differ fundamentally in their interactions with HSP70 chaperones. Using nuclear magnetic resonance spectroscopy6,7 we find that the major class B JDPs are regulated by an autoinhibitory mechanism that is not present in other classes. Although in all JDPs the interaction of the characteristic J-domain is responsible for the activation of HSP70, in DNAJB1 the HSP70-binding sites in this domain are intrinsically blocked by an adjacent glycine-phenylalanine rich region—an inhibition that can be released upon the interaction of a second site on DNAJB1 with the HSP70 C-terminal tail. This regulation, which controls substrate targeting to HSP70, is essential for the disaggregation of amyloid fibres by HSP70–DNAJB1, illustrating why no other class of JDPs can substitute for class B in this function. Moreover, this regulatory layer, which governs the functional specificities of JDP co-chaperones and their interactions with HSP70s, could be key to the wide range of cellular functions of HSP70.

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Fig. 1: The GF region of class B JDPs initially blocks J-domain binding to HSP70.
Fig. 2: DNAJB1 contains an additional HSP70-binding site that is not found in class A JDPs.
Fig. 3: DNAJB1 binding to the C-terminal EEVD tail of HSP70 releases the JD–GF inhibition.
Fig. 4: DNAJB1 JD–GF inhibition is essential for amyloid disaggregation.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. NMR chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank under the following accession codes: 50169 for DNAJB1JD, 50168 for DNAJA2JD and 50167 for DNAJB1JD–GF. The structure of DNAJB1JD–GF has been deposited to the Protein Data Bank (PDB) under accession code 6Z5N. Source data are provided with this paper.

References

  1. Mayer, M. P. & Bukau, B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670–684 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kampinga, H. H. & Craig, E. A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol. 11, 579–592 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Rosenzweig, R., Nillegoda, N. B., Mayer, M. P. & Bukau, B. The Hsp70 chaperone network. Nat. Rev. Mol. Cell Biol. 20, 665–680 (2019).

    CAS  PubMed  Google Scholar 

  4. Tiwari, S., Kumar, V., Jayaraj, G. G., Maiti, S. & Mapa, K. Unique structural modulation of a non-native substrate by cochaperone DnaJ. Biochemistry 52, 1011–1018 (2013).

    CAS  PubMed  Google Scholar 

  5. Jiang, Y., Rossi, P. & Kalodimos, C. G. Structural basis for client recognition and activity of Hsp40 chaperones. Science 365, 1313–1319 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tugarinov, V., Hwang, P. M., Ollerenshaw, J. E. & Kay, L. E. Cross-correlated relaxation enhanced 1H–13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J. Am. Chem. Soc. 125, 10420–10428 (2003).

    CAS  PubMed  Google Scholar 

  7. Tugarinov, V., Kanelis, V. & Kay, L. E. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat. Protoc. 1, 749–754 (2006).

    CAS  PubMed  Google Scholar 

  8. Kityk, R., Kopp, J. & Mayer, M. P. Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones. Mol. Cell 69, 227–237.e4, (2018).

    CAS  PubMed  Google Scholar 

  9. Wentink, A., Nussbaum-Krammer, C. & Bukau, B. Modulation of amyloid states by molecular chaperones. Cold Spring Harb. Perspect. Biol. 11, a033969 (2019).

    CAS  PubMed  Google Scholar 

  10. Schilke, B. A. et al. Broadening the functionality of a J-protein/HSP70 molecular chaperone system. PLoS Genet. 13, e1007084 (2017).

    PubMed  PubMed Central  Google Scholar 

  11. Zarouchlioti, C., Parfitt, D. A., Li, W., Gittings, L. M. & Cheetham, M. E. DNAJ proteins in neurodegeneration: essential and protective factors. Phil. Trans. R. Soc. Lond. B 373, 20160534 (2018).

    Google Scholar 

  12. Gao, X. et al. Human HSP70 disaggregase reverses Parkinson’s-linked α-synuclein amyloid fibrils. Mol. Cell 59, 781–793 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Rampelt, H. et al. Metazoan HSP70 machines use HSP110 to power protein disaggregation. EMBO J. 31, 4221–4235 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Duennwald, M. L., Echeverria, A. & Shorter, J. Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans. PLoS Biol. 10, e1001346 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Scior, A. et al. Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex. EMBO J. 37, 282–299 (2018).

    CAS  PubMed  Google Scholar 

  16. Nachman, E. et al. Disassembly of Tau fibrils by the human Hsp70 disaggregationmachinery generates small seeding-competent species. Preprint at https://doi.org/10.1101/2019.12.16.876888 (2019).

  17. Minami, Y., Höhfeld, J., Ohtsuka, K. & Hartl, F. U. Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. J. Biol. Chem. 271, 19617–19624 (1996).

    CAS  PubMed  Google Scholar 

  18. Karzai, A. W. & McMacken, R. A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271, 11236–11246 (1996).

    CAS  PubMed  Google Scholar 

  19. Yan, W. & Craig, E. A. The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1. Mol. Cell. Biol. 19, 7751–7758 (1999). │

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Shen, Y. & Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227–241 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Mesleh, M. F., Veglia, G., DeSilva, T. M., Marassi, F. M. & Opella, S. J. Dipolar waves as NMR maps of protein structure. J. Am. Chem. Soc. 124, 4206–4207 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Qian, Y. Q., Patel, D., Hartl, F. U. & McColl, D. J. Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain. J. Mol. Biol. 260, 224–235 (1996).

    CAS  PubMed  Google Scholar 

  23. Yu, H. Y. et al. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1. J. Mol. Biol. 427, 1632–1643 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, J., Wu, Y., Qian, X. & Sha, B. Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex. Biochem. J. 398, 353–360 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Craig, E. A. & Marszalek, J. How do J-proteins get Hsp70 to do so many different things? Trends Biochem. Sci. 42, 355–368 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Yu, H. Y., Ziegelhoffer, T. & Craig, E. A. Functionality of class A and class B J-protein co-chaperones with Hsp70. FEBS Lett. 589, 2825–2830 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Battiste, J. L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39, 5355–5365 (2000).

    CAS  PubMed  Google Scholar 

  28. Clore, G. M. & Iwahara, J. Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem. Rev. 109, 4108–4139 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Suzuki, H. et al. Peptide-binding sites as revealed by the crystal structures of the human Hsp40 Hdj1 C-terminal domain in complex with the octapeptide from human Hsp70. Biochemistry 49, 8577–8584 (2010).

    CAS  PubMed  Google Scholar 

  30. Wentink, A. S. et al. Molecular dissection of amyloid disaggregation by human HSP70. Nature https://doi.org/10.1038/s41586-020-2904-6 (2020).

  31. Karamanos, T. K., Tugarinov, V. & Clore, G. M. Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis. Proc. Natl Acad. Sci. USA 116, 21529–21538 (2019).

    CAS  PubMed  Google Scholar 

  32. Ruggieri, A. et al. Complete loss of the DNAJB6 G/F domain and novel missense mutations cause distal-onset DNAJB6 myopathy. Acta Neuropathol. Commun. 3, 44 (2015).

    PubMed  PubMed Central  Google Scholar 

  33. Shen, Y. & Hendershot, L. M. ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP’s interactions with unfolded substrates. Mol. Biol. Cell 16, 40–50 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Gelis, I. et al. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131, 756–769 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Gans, P. et al. Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high-molecular-weight proteins. Angew. Chem. Int. Ed. 49, 1958–1962 (2010).

    CAS  Google Scholar 

  36. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Google Scholar 

  37. Goddard, T. D. & Kneller, D. G. SPARKY 3 (University of California, San Francisco, 2002).

  38. Vranken, W. F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 (2005).

    CAS  PubMed  Google Scholar 

  39. Karagöz, G. E. et al. N-terminal domain of human Hsp90 triggers binding to the cochaperone p23. Proc. Natl Acad. Sci. USA 108, 580–585 (2011).

    ADS  PubMed  Google Scholar 

  40. Sekhar, A., Rosenzweig, R., Bouvignies, G. & Kay, L. E. Mapping the conformation of a client protein through the Hsp70 functional cycle. Proc. Natl Acad. Sci. USA 112, 10395–10400 (2015).

    ADS  CAS  PubMed  Google Scholar 

  41. Constantine, K. L. et al. Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy. J. Biomol. NMR 3, 41–54 (1993).

    CAS  PubMed  Google Scholar 

  42. Farrow, N. A. et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

    CAS  PubMed  Google Scholar 

  43. Lyons, B. A., Tashiro, M., Cedergren, L., Nilsson, B. & Montelione, G. T. An improved strategy for determining resonance assignments for isotopically enriched proteins and its application to an engineered domain of staphylococcal protein A. Biochemistry 32, 7839–7845 (1993).

    CAS  PubMed  Google Scholar 

  44. Logan, T. M., Olejniczak, E. T., Xu, R. X. & Fesik, S. W. A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments. J. Biomol. NMR 3, 225–231 (1993).

    CAS  PubMed  Google Scholar 

  45. Yamazaki, T., Formankay, J. D. & Kay, L. E. Two-dimensional NMR experiments for correlating 13Cβ and 1Hδ/ε chemical shifts of aromatic residues in 13C-labeled proteins via scalar couplings. J. Am. Chem. Soc. 115, 11054–11055 (1993).

    CAS  Google Scholar 

  46. Tugarinov, V., Choy, W. Y., Orekhov, V. Y. & Kay, L. E. Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proc. Natl Acad. Sci. USA 102, 622–627 (2005).

    ADS  CAS  PubMed  Google Scholar 

  47. Zwahlen, C. et al. An NMR experiment for measuring methyl–methyl NOEs in 13C-labeled proteins with high resolution. J. Am. Chem. Soc. 120, 7617–7625 (1998).

    CAS  Google Scholar 

  48. Lakomek, N. A., Ying, J. & Bax, A. Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods. J. Biomol. NMR 53, 209–221 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Mandel, A. M., Akke, M. & Palmer, A. G., III. Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J. Mol. Biol. 246, 144–163 (1995).

    CAS  PubMed  Google Scholar 

  50. Press, W. H., Teukolsky, S. A., Vetterling, W. T. & Flannery, B. P. Numerical Recipes in C: The Art of Scientific Computing 2nd edn (Cambridge Univ. Press, 1992).

  51. Ottiger, M., Delaglio, F. & Bax, A. Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J. Magn. Reson. 131, 373–378 (1998).

    ADS  CAS  PubMed  Google Scholar 

  52. Shen, Y. et al. Consistent blind protein structure generation from NMR chemical shift data. Proc. Natl Acad. Sci. USA 105, 4685–4690 (2008).

    ADS  CAS  PubMed  Google Scholar 

  53. Shen, Y., Vernon, R., Baker, D. & Bax, A. De novo protein structure generation from incomplete chemical shift assignments. J. Biomol. NMR 43, 63–78 (2009).

    CAS  PubMed  Google Scholar 

  54. Kuenze, G., Bonneau, R., Leman, J. K. & Meiler, J. Integrative protein modeling in RosettaNMR from sparse paramagnetic restraints. Structure 27, 1721–1734.e5 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Vernon, R., Shen, Y., Baker, D. & Lange, O. F. Improved chemical shift based fragment selection for CS-Rosetta using Rosetta3 fragment picker. J. Biomol. NMR 57, 117–127 (2013).

    CAS  PubMed  Google Scholar 

  56. Shen, Y. & Bax, A. SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network. J. Biomol. NMR 48, 13–22 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Bhattacharya, A., Tejero, R. & Montelione, G. T. Evaluating protein structures determined by structural genomics consortia. Proteins 66, 778–795 (2007).

    CAS  PubMed  Google Scholar 

  58. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    CAS  PubMed  Google Scholar 

  59. Nillegoda, N. B. et al. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524, 247–251 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. Tsai, J. & Douglas, M. G. A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. J. Biol. Chem. 271, 9347–9354 (1996).

    CAS  PubMed  Google Scholar 

  61. Suh, W. C., Lu, C. Z. & Gross, C. A. Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ. J. Biol. Chem. 274, 30534–30539 (1999).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank T. Scherf for NMR support and the Clore Institute for High-Field Magnetic Resonance Imaging and Spectroscopy; and D. Fass for discussions and advice. R.R. is supported by the European Research Council starting grant (ERC-2018-STG 802001), the Minerva Foundation, and a research grant from the Blythe Brenden-Mann New Scientist Fund. B.B. is supported by the Deutsche Forschungsgemeinschaft grant (SFB 1036, BU617/19-1) and the Helmholtz-Gemeinschaft (German-Israeli Helmholtz Research School in Cancer; AmPro) to B.B. and R.R. M.M. acknowledges the support of the Helmholtz International Graduate School for Cancer Research at the DKFZ. N.L. is the incumbent of the Alan and Laraine Fischer Career Development Chair, and is supported by the Israel Science Foundation (grant no. 2462/19).

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Author notes

  1. These authors contributed equally: Ofrah Faust, Meital Abayev-Avraham

Authors and Affiliations

  1. Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

    Ofrah Faust, Meital Abayev-Avraham, Michael Maurer & Rina Rosenzweig

  2. Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany

    Anne S. Wentink, Michael Maurer, Nadinath B. Nillegoda & Bernd Bukau

  3. Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Victoria, Australia

    Nadinath B. Nillegoda

  4. Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

    Nir London

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  1. Ofrah Faust
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Contributions

O.F., M.A.-A., A.S.W., N.B.N. and R.R. designed the research; O.F., M.A.-A. and M.M. performed the NMR spectroscopy measurements, processed and analysed the data; M.A.-A. determined the NMR structure of DNAJB1JD–GF together with N.L. and R.R.; O.F., A.S.W., N.B.N. and M.M. performed the biochemical and functional assays; O.F., M.A.-A., A.S.W., M.M., N.B.N., B.B. and R.R. analysed data; and O.F., M.A.-A., M.M., A.S.W., N.B.N., B.B. and R.R. wrote the paper.

Corresponding authors

Correspondence to Bernd Bukau or Rina Rosenzweig.

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Extended data figures and tables

Extended Data Fig. 1 Interaction of JDP J-domains with HSP70 chaperone.

a–c, Residue-resolved NMR signal intensity ratios I/I0, where I and I0 are signal intensities for HSP70-bound and free DNAJA2JD (a), DNAJB1JD (b), and DNAJA1JD (c), respectively. The positions of the four helices in each J-domain are indicated at the bottom of the plot. Large changes in intensity are detected at the end of helix II, the flexible loop containing the conserved HPD motif, and at helix III, corresponding to HSP70-binding sites. In all three J-domains, similar residues are involved in binding, pointing to high conservation of the JD–HSP70 interaction. d, Overlay of 1H–15N HSQC spectra of 0.2 mM DNAJA1JD–GF alone (in black), and in the presence of twofold excess of protonated (1H) HSP70 (green). Upon complex formation with HSP70, the majority of the J-domain peaks were broadened out beyond detection, whereas the GF residues, that are part of a flexible disordered linker, were not affected, indicating that only the J-domain—and not the GF-rich region—interacts with the HSP70 chaperone. Assignments of J-domain residues are indicated on the spectrum. e, 1H–15N HSQC spectra of 0.2 mM DNAJB1JD alone (in black), and in the presence of twofold excess protonated (1H) HSP70 (red). Here too, addition of protonated HSP70 causes severe peak broadening in the majority of the J-domain residues, with the exception of the flexible terminal residues and side chains. This, however, is in contrast to the spectrum of DNAJB1JD–GF under the same conditions (Fig. 1f), which showed no peak broadening, indicating lack of interaction with HSP70. f, Comparison of chemical shifts of residues 1–71 in the DNAJB1JD and the same residues in the DNAJB1JD–GF construct. The differences in the chemical shift positions (Δδ) are defined by the relation \(\Delta \delta =\sqrt{{(\Delta {\delta }_{{\rm{H}}})}^{2}+{\left(\frac{\Delta {\delta }_{{\rm{N}}}}{5}\right)}^{2}}\), where ΔδH and ΔδN are the 1H proton and 15N nitrogen shift changes between the chemical shifts of DNAJB1JD and DNAJB1JD–GF residues (1–71). Large chemical shift changes are seen in residues of helices II and III, which are coloured red on the structure of the DNAJB1 J-domain. Residues displaying large differences between DNAJB1JD and DNAJB1JD–GF are the same ones that show changes upon HSP70 binding (helices II and III), and those in helix IV.

Extended Data Fig. 2 Structural characterization of DNAJB1JD–GF.

a, TALOS+ secondary structure probabilities20 of DNAJB1JD–GF derived from backbone 13Cα, 13Cβ, 13C′, 15N and 1HN chemical shifts. Predicted α-helices are shown in red and β-sheets in blue. b, Backbone order parameters (S2) calculated using ModelFree49 from 15N relaxation data (see Methods). The location of the five helices in DNAJB1JD–GF and their boundaries is indicated above the plot. c, Backbone amide RDCs (1DNH) collected in 16 mg ml−1 phage pf1 alignment media. d–f, 1HN PRE (R2ox–R2red) profiles recorded for nitroxide spin-labelled DNAJB1JD–GF with the MTSL spin-label located at residues 108 (d), 56 (e) and 40 (f). 1H–15N cross-peaks that are broadened beyond detection in the oxidized state and were calculated to have a PRE ≥ 200 (see Methods) are shown as grey bars. g, Correlation between observed RDCs in pf1 alignment media and those calculated for the 10 lowest energy CS-Rosetta52,53,54 structures. The RDC R-factor, Rdip, is given by: \({R}_{{\rm{dip}}}={\{\langle {({D}_{{\rm{obs}}}-{D}_{{\rm{cal}}})}^{2}\rangle /(2\langle {{D}_{{\rm{obs}}}}^{2}\rangle )\}}^{\frac{1}{2}}\) where Dobs and Dcal are the observed and calculated RDC values, respectively. Da and η are the magnitude (normalized to the N–H RDCs) and rhombicity, respectively, of the alignment tensor. h, Select strips from a 3D NOESY–HSQC experiment depicting long range methyl-NH NOEs for residues 28–102 (left), 29–106 (middle) and 101–56 (right), and indicating long range interactions between helix V and helices II and III.

Extended Data Fig. 3 Deletion of helix V removes the GF inhibition of the J-domain and restores HSP70 binding.

a, Overlay of 1H–15N HSQC correlation maps of 0.2 mM DNAJB11–96 alone (black), and in complex with 0.2 mM HSP70 (red). Deletion of helix V residues restores the ability of the DNAJB1 J-domain to interact with HSP70, even in the presence of the GF region. b, Differences in chemical shifts between DNAJB1JD (residues 1–71) and the DNAJB11–96 construct lacking helix V. No substantial changes in the spectra were observed between the two constructs, with the exception of residues located in helix IV (coloured red on the structure of the DNAJB1 J-domain), which connects the J-domain and the GF region. c, 1H–13C HMQC spectra showing the leucine/valine (left) and isoleucine (right) regions of 0.2 mM 13CH3-ILVM labelled full-length DNAJB1 (grey), DNAJB1JD (red) and DNAJB1JD–GF (orange). The DNAJB1JD–GF peaks overlap with those of full-length DNAJB1 (with the exception of small chemical-shift perturbations observed for residues 29 and 48), whereas those of the free J-domain do not, indicating that in the full-length protein the J-domain is in the GF-inhibited conformation.

Extended Data Fig. 4 Interaction of DNAJB1 with HSP70.

a, Chemical-shift perturbations induced by HSP70 binding to [2H, 13CH3-ILVM]-labelled DNAJB1. Chemical-shift perturbations are defined by the relation \(\Delta \delta =\sqrt{{\left(\frac{\Delta {\delta }_{{\rm{H}}}}{\alpha }\right)}^{2}+{\left(\frac{\Delta {\delta }_{{\rm{C}}}}{\beta }\right)}^{2}}\), where ΔδH and ΔδC are methyl 1H and 13C chemical shift changes between apo and bound forms of the protein, and α (β) is one standard deviation from the methyl 1H (13C) chemical shifts deposited in the Biological Magnetic Resonance Data Bank (α is 0.29 (I), 0.28 (L), 0.27 (V) and 0.41 (M), whereas β is 1.65 (I), 1.6 (L), 1.4 (V), and 1.54 (M)). Whereas HSP70 binds to two sites in DNAJB1 (J-domain and CTDI), large chemical shifts changes are observed only in CTDI, indicating that this domain binds to HSP70 in a transient manner (on a fast NMR timescale). b, Per-residue peak intensity ratios of DNAJB1(32QPN34) in the absence and presence of HSP70 chaperone. In this variant, conserved residues (32–34) in the J-domain HPD motif, which is essential for HSP70 activation, were mutated to QPN. Upon addition of HSP70, no changes in intensities were detected for the J-domain residues, indicating that—as expected—mutations to the HPD motif abolish the JD–HSP70 interaction60,61. The DNAJB1 CTDI interaction with HSP70, however, remained strong and was unaffected by the mutation. c, Peak intensity ratios of DNAJB1 CTDs (residues 154–340) alone, and in complex with HSP70 chaperone. The interaction of HSP70 with the CTDI of DNAJB1 occurs independently of the J-domain. d, Differences in chemical shifts between free DNAJB1 and DNAJB1 in complex with a synthetic peptide corresponding to the last 20 amino acids of the HSP70 C-terminal tail. Large chemical shift perturbations are detected in the CTDI region of DNAJB1, similar to those observed upon DNAJB1 binding to full-length HSP70 (see a).

Extended Data Fig. 5 HSP70 binds to the CTDI of DNAJB1 but not of DNAJA2.

a–c, Overlay of  1H–13C HMQC correlation maps of ILVM-labelled HSP70 (black) and HSP70 in complex with DNAJB1 (a, light red), DNAJB1 J-domain (b, dark red), and DNAJB1 CTDs (c, cyan). The DNAJB1 J-domain interacts with residues in the HSP70 nucleotide-binding domain and substrate-binding domain, similar to the interaction observed between E. coli DnaK and the J-domain of DnaJ8. DNAJB1 CTDs, however, interact with a different region of HSP70, corresponding to the disordered C-terminal tail of HSP70. d–f, Interaction of ILVM-labelled HSP70 with DNAJA2 (d, light blue), DNAJA2 J-domain (e, dark blue) and DNAJA2 CTDs (f, green). The residues of the DNAJA2 J-domain interact with similar HSP70 residues to the DNAJB1 J-domain, in both cases located at the interface between the nucleotide-binding domain and the substrate-binding domain. Unlike DNAJB1, however, no interaction was observed between the DNAJA2 CTDs and HSP70.

Extended Data Fig. 6 Client proteins and the HSP70 C-terminal EEVD tail bind simultaneously to DNAJB1.

a, Per-residue peak intensity ratios of DNAJB1 in complex with excess α-synuclein over DNAJB1 alone. Lower ratios indicate greater changes between bound and free states. b, Residues displaying substantial changes in intensities (2 standard deviations below mean) are shown as yellow spheres on the structure of DNAJB1 CTDs (PDB ID: 3AGX29). α-synuclein interacts predominantly with the CTDII domain of the chaperone, with only minor changes being observed in CTDI residues. c, d, Competition experiments between α-synuclein and a synthetic peptide corresponding to the last 20 amino acids in the HSP70 C-terminal tail (EEVD peptide). DNAJB1 residues showing broadening upon interaction with α-synuclein are unaffected by addition of the EEVD peptide (c). Likewise, the behaviour of DNAJB1 residues displaying shifts upon EEVD peptide binding was unchanged in the presence of α-synuclein (d), indicating that DNAJB1 can interact simultaneously with both.

Extended Data Fig. 7 Removal of the HSP70 EEVD tail abolishes binding to class B JDPs.

a, b, 1H–13C HMQC spectra of HSP70 (pink) and HSP70(ΔEEVD) (black) showing the isoleucine (a) and valine (b) spectral regions. No conformational changes were detected in the HSP70 chaperone upon deletion of the four C-terminal residues (EEVD). The only visible difference between the two spectra is the lack of the V645 peak in b, as this residue is part of the C-terminal tail that was deleted in HSP70ΔEEVD. c, d, Interaction of HSP70(ΔEEVD) with DNAJA2 (c) and DNAJB1 (d). The interaction between DNAJA2 and HSP70(ΔEEVD) occurs via the same residues as between DNAJA2 and wild-type HSP70 (c, blue; compare to Extended Data Fig. 5c), whereas no interaction was detected between HSP70(ΔEEVD) and DNAJB1 (d, red).

Extended Data Fig. 8 DNAJB1 mutants with released GF inhibition of the J-domain.

a, b, Cartoon representation of DNAJB1JD–GF, highlighting the position of residues E50 (a) and F102 (b) that were mutated to alanines. The F102A mutation was designed on the basis of our DNAJB1JD–GF structure to disrupt the hydrophobic contacts between J-domain residues A28, L29, F45 and helix V. The E50A mutation was previously reported (in Sis1) to rescue the defects in protein refolding activity of HSP70(ΔEEVD)23,26 and we therefore suspected it also released the J-domain. c, d, 1H-15N HSQC spectra of DNAJB1JD–GF(E50A) (c) and DNAJB1JD–GF(F102A) (d) alone, and in complex with HSP70 (teal). Whereas wild-type DNAJB1JD–GF was unable to bind HSP70, partial release of the J-domain by targeted mutation in either the J-domain or the GF region generates DNAJB1JD–GF constructs that bind HSP70 with high affinity—as indicated by the severe broadening of the peaks in the bound spectrum. e, f, Selected regions of the HSQC spectra showing residues D57 and D65 of DNAJB1JD–GF(E50A) (e) and DNAJB1JD–GF(F102A) (f) in slow exchange between the GF-inhibited and released J-domain conformations. The population of each conformation was calculated by integrating the peak volumes, and is indicated for each residue. Overall,  the E50A mutation to the J-domain results in an equilibrium in which 64% of all J-domain residues are in a released conformation,  whereas in the F102A mutation to the GF helix V, 36% are released.

Extended Data Fig. 9 DNAJB1 mutants with partially released J-domains can interact with HSP70 lacking the C-terminal tail.

a–c, Peak intensity ratios of wild-type DNAJB1 (a), DNAJB1(F102A) (b) and DNAJB1(E50A) (c) in the presence of twofold molar excess of HSP70(ΔEEVD) chaperone lacking the four last residues (EEVD) of the C-terminal tail. All plots are normalized, per residue, by the peak intensities of each of the DNAJB1 variants alone. Wild-type DNAJB1 protein is unable to bind to HSP70(ΔEEVD), as HSP70 C-terminal is required for the release of its J-domain. However, both DNAJB1 mutants with partially released J-domains bound to HSP70(ΔEEVD), though the interaction was only through their J-domain, as the presence of the HSP70 C-terminal tail is required for binding of DNAJB1 CTDI (e, f).

Extended Data Fig. 10 Characterization of HSP70 activity with constitutively JD-released (DNAJB1(ΔH5)) mutant.

a, Spectrum of DNAJB1(ΔH5) mutant (light purple), containing a released J-domain, overlaid onto the spectrum of wild-type DNAJB1(dark purple). Outside of the J-domain residues, no changes were observed to the overall conformation of the chaperone. b, DNAJB1 J-domain residues L55 and V54 display different chemical shifts in the GF-inhibited (grey) and free J-domain (black) conformations. The J-domain of the DNAJB1(ΔH5) mutant is shown to be fully released. c, d, The steady-state ATPase activity of 250 nM HSP70 (c) or HSP70(ΔEEVD) (d) chaperone measured in the presence of increasing concentrations of DNAJA2 (blue), wild-type DNAJB1 (red), DNAJB1(E50A) (orange), DNAJB1(F102A) (yellow) or DNAJB1(ΔH5) (purple) variants. Experiments were performed at 0, 2.5, 10, 50, 125, 250 and 500 nM, with each concentration being shown as a separate bar representing the mean and s.d. of three experiments, normalized to the activity of HSP70 alone. e, Fluorescence anisotropy measurements of DNAJA2 (blue), DNAJB1 (red) and DNAJB1(ΔH5) (purple) binding to preformed α-synuclein fibrils. Data points represent the mean and s.d. of 3 independent measurements, with the Kd values for DNAJA2, DNAJB1 and DNAJB1(ΔH5) being 1,150 ± 170 nM, 780 ± 38 nM and 730 ± 50 nM, respectively. f, Fluorescence anisotropy measurements of DNAJB1 (red) and DNAJB1(ΔH5) (purple) binding to the HSP70(T204A) ATPase-deficient mutant. Data points represent the mean and s.d. of 3 independent measurement, with apparent Kd values for DNAJB1 and DNAJB1(ΔH5) being 10.6 ± 2.3 μM and 5.4 ± 1.4 μM, respectively. g, Co-sedimentation assay assessing the recruitment of HSP70 to preformed α-synuclein fibres by DNAJA2, DNAJB1 and DNAJB1(ΔH5) chaperones. The assay was performed with 5 μM (1×) and 50 μM (10×) of the indicated JDPs. Both DNAJB1 and DNAJB1(ΔH5) efficiently recruit HSP70 to the fibres, whereas DNAJA2—which has a single HSP70-binding site per protomer—does not. The experiment was repeated twice with similar results. h, Recruitment of HSP70 to α-synuclein fibres by DNAJA2 (blue), DNAJB1 (red) and DNAJB1(ΔH5) (purple) measured by fluorescence anisotropy. Data points represent the mean and s.d. of 3 independent measurements. i, Relative FRET efficiencies measured for wild-type HSP70 or HSP70(ΔEEVD) incubated with α-synuclein fibres and in the presence of DNAJA2 (blue), wild-type DNAJB1 (red) or DNAJB1(ΔH5) (purple). Data are presented as mean ± s.e.m. of 3 independent measurements. In each experiment, half of the HSP70 population was labelled with C494-AF488 (donor) and half with C494-AF584 (acceptor), and a FRET signal was observed when donor- and acceptor-labelled HSP70s were in close proximity, indicating clustering.

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The file contains Supplementary Table 1 and Supplementary Fig. 1. Supplementary Table 1 - NMR and refinement statistics for DnaJB1JD-GF NMR structure. Supplementary Fig. 1 | Gel source data for Extended Data Fig. 10g. The content of Extended Data Fig. 10g is highlighted in a box. The experiment was repeated two times with similar results.

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Faust, O., Abayev-Avraham, M., Wentink, A.S. et al. HSP40 proteins use class-specific regulation to drive HSP70 functional diversity. Nature 587, 489–494 (2020). https://doi.org/10.1038/s41586-020-2906-4

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