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Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2

Nature Structural & Molecular Biology volume 15, pages 1309–1317 (2008)Cite this article

Abstract

Cochaperones are essential for Hsp70- and Hsc70-mediated folding of proteins and include nucleotide-exchange factors (NEFs) that assist protein folding by accelerating ADP-ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as an NEF, but the molecular basis for its function is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the 'brand new bag' (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure, in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client binding sites and Hsc70-interaction sites of the Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp70-mediated protein folding by Bag2.

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Figure 1: Structure of the Bag2-BNB domain.
Figure 2: Structure of the Hsc70-NBD–Bag2-BNB domain complex.
Figure 3: Mechanism of Bag2-BNB–mediated nucleotide exhange.
Figure 4: Effects of Bag2 mutations on Hsc70 binding, nucleotide exchange and substrate refolding activity.
Figure 5: Mapping of chaperone client binding sites on Bag2-BNB domains.
Figure 6: Models for Hsp70, Bag2 and chaperone client interaction.

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Acknowledgements

This work was funded by grants from the US National Institutes of Health (R01-GM61728 to C.P. and RO1-GM080271 to S.M.), the American Heart Association (SDG 0735313N to S.M.) and by funds from the State of Ohio Eminent Scholar Program (to A.B.H.). E.K. was supported by funds from the Ralph Wilson Medical Research Foundation. The Advanced Light Source is supported by the US Department of Energy under contract number DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

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Author notes

  1. Richard C Page, Michelle M Gomes and Ekta Kohli: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Cardiology, Lerner Research Institute, NB50, 9500 Euclid Avenue, The Cleveland Clinic, Cleveland, 44195, Ohio, USA

    Zhen Xu, Richard C Page, Ekta Kohli & Saurav Misra

  2. Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, 45267, Ohio, USA

    Michelle M Gomes & Andrew B Herr

  3. Molecular Biology Consortium, Beamline 4.2.2, Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, 94720, California, USA

    Jay C Nix

  4. Carolina Cardiovascular Biology Center and Departments of Pharmacology, Cell and Developmental Biology, and Medicine, University of North Carolina, CB#7075, Burnett-Womack Building, 099 Manning Drive, Chapel Hill, 27599, North Carolina, USA

    Cam Patterson

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Contributions

Z.X. and E.K. cloned, expressed and purified the various protein constructs; Z.X., J.C.N. and S.M. carried out crystallization, data collection and crystal structure solution; R.C.P. expressed and purified labeled proteins and carried out NMR experiments and data analysis; M.M.G. carried out analytical ultracentrifugation experiments, and M.M.G. and A.B.H. interpreted the ultracentrifugation data; E.K. and Z.X. carried out in vitro protein-protein interaction and Luciferase-refolding experiments; C.P. performed the single-turnover nucleotide-exchange assays; S.M. compiled the manuscript with contributions from Z.X., R.C.P., E.K., A.B.H. and C.P.

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Correspondence to Saurav Misra.

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Xu, Z., Page, R., Gomes, M. et al. Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2. Nat Struct Mol Biol 15, 1309–1317 (2008). https://doi.org/10.1038/nsmb.1518

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