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Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions

Abstract

The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain–binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered CH1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

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Figure 1: Kinetics and thermodynamics for the interaction of BiP with the CH1 domain and the CH1-derived HTFPAVL peptide.
Figure 2: Labeling positions within BiP for spFRET measurements.
Figure 3: Single-pair FRET analysis of BiP 166/518.
Figure 4: Single-pair FRET analysis of BiP 518/636.
Figure 5: BiP and substrate binding by ERdJ3 and its influence on BiP conformation.
Figure 6: Influence of ERdJ3 on substrate binding by BiP.
Figure 7: Model for the ERdJ3-regulated chaperone cycle of BiP.

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References

  1. Chen, Y. et al. SPD—a web-based secreted protein database. Nucleic Acids Res. 33, D169–D173 (2005).

    CAS  Article  PubMed  Google Scholar 

  2. Hebert, D.N. & Molinari, M. In and out of the ER: protein folding, quality control, degradation, and related human diseases. Physiol. Rev. 87, 1377–1408 (2007).

    CAS  Article  PubMed  Google Scholar 

  3. Munro, S. & Pelham, H.R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46, 291–300 (1986).

    CAS  Article  PubMed  Google Scholar 

  4. Karlin, S. & Brocchieri, L. Heat shock protein 70 family: multiple sequence comparisons, function, and evolution. J. Mol. Evol. 47, 565–577 (1998).

    CAS  Article  PubMed  Google Scholar 

  5. Haas, I.G. & Wabl, M. Immunoglobulin heavy chain binding protein. Nature 306, 387–389 (1983).

    CAS  Article  PubMed  Google Scholar 

  6. Weitzmann, A., Baldes, C., Dudek, J. & Zimmermann, R. The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum—a quantitative approach. FEBS J. 274, 5175–5187 (2007).

    CAS  Article  PubMed  Google Scholar 

  7. Kozutsumi, Y., Segal, M., Normington, K., Gething, M.J. & Sambrook, J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332, 462–464 (1988).

    CAS  Article  PubMed  Google Scholar 

  8. Kassenbrock, C.K., Garcia, P.D., Walter, P. & Kelly, R.B. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333, 90–93 (1988).

    CAS  Article  PubMed  Google Scholar 

  9. Alder, N.N., Shen, Y., Brodsky, J.L., Hendershot, L.M. & Johnson, A.E. The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum. J. Cell Biol. 168, 389–399 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Dudek, J. et al. ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum. Nat. Struct. Mol. Biol. 12, 1008–1014 (2005).

    CAS  Article  PubMed  Google Scholar 

  11. Otero, J.H., Lizak, B. & Hendershot, L.M. Life and death of a BiP substrate. Semin. Cell Dev. Biol. 21, 472–478 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Vembar, S.S. & Brodsky, J.L. One step at a time: endoplasmic reticulum-associated degradation. Nat. Rev. Mol. Cell Biol. 9, 944–957 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Bole, D.G., Hendershot, L.M. & Kearney, J.F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J. Cell Biol. 102, 1558–1566 (1986).

    CAS  Article  PubMed  Google Scholar 

  14. Feige, M.J. et al. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol. Cell 34, 569–579 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Lee, Y.K., Brewer, J.W., Hellman, R. & Hendershot, L.M. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Mol. Biol. Cell 10, 2209–2219 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Blond-Elguindi, S. et al. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75, 717–728 (1993).

    CAS  Article  PubMed  Google Scholar 

  17. Knarr, G., Gething, M.J., Modrow, S. & Buchner, J. BiP binding sequences in antibodies. J. Biol. Chem. 270, 27589–27594 (1995).

    CAS  Article  PubMed  Google Scholar 

  18. Gething, M.J. et al. Binding sites for Hsp70 molecular chaperones in natural proteins. Cold Spring Harb. Symp. Quant. Biol. 60, 417–428 (1995).

    CAS  Article  PubMed  Google Scholar 

  19. Knarr, G., Modrow, S., Todd, A., Gething, M.J. & Buchner, J. BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip. J. Biol. Chem. 274, 29850–29857 (1999).

    CAS  Article  PubMed  Google Scholar 

  20. Rüdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Bukau, B., Weissman, J. & Horwich, A. Molecular chaperones and protein quality control. Cell 125, 443–451 (2006).

    CAS  Article  PubMed  Google Scholar 

  22. Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606–1614 (1996).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Swain, J.F. et al. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. Mol. Cell 26, 27–39 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Bertelsen, E.B., Chang, L., Gestwicki, J.E. & Zuiderweg, E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc. Natl. Acad. Sci. USA 106, 8471–8476 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Vogel, M., Mayer, M.P. & Bukau, B. Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker. J. Biol. Chem. 281, 38705–38711 (2006).

    CAS  Article  PubMed  Google Scholar 

  26. Goloubinoff, P. & De Los, R.P. The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem. Sci. 32, 372–380 (2007).

    CAS  Article  PubMed  Google Scholar 

  27. Jiang, J., Lafer, E.M. & Sousa, R. Crystallization of a functionally intact Hsc70 chaperone. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 39–43 (2006).

    CAS  Article  PubMed  Google Scholar 

  28. Mapa, K. et al. The conformational dynamics of the mitochondrial Hsp70 chaperone. Mol. Cell 38, 89–100 (2010).

    CAS  Article  PubMed  Google Scholar 

  29. Woo, H.J., Jiang, J., Lafer, E.M. & Sousa, R. ATP-induced conformational changes in Hsp70: molecular dynamics and experimental validation of an in silico predicted conformation. Biochemistry 48, 11470–11477 (2009).

    CAS  Article  PubMed  Google Scholar 

  30. Craig, E.A., Huang, P., Aron, R. & Andrew, A. The diverse roles of J-proteins, the obligate Hsp70 co-chaperone. Rev. Physiol. Biochem. Pharmacol. 156, 1–21 (2006).

    CAS  PubMed  Google Scholar 

  31. 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  Article  PubMed  PubMed Central  Google Scholar 

  32. Meunier, L., Usherwood, Y.K., Chung, K.T. & Hendershot, L.M. A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins. Mol. Biol. Cell 13, 4456–4469 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Jin, Y., Zhuang, M. & Hendershot, L.M. ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues. Biochemistry 48, 41–49 (2009).

    CAS  Article  PubMed  Google Scholar 

  34. Jin, Y., Awad, W., Petrova, K. & Hendershot, L.M. Regulated release of ERdj3 from unfolded proteins by BiP. EMBO J. 27, 2873–2882 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 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  Article  PubMed  PubMed Central  Google Scholar 

  36. Bies, C. et al. Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p. Biol. Chem. 385, 389–395 (2004).

    CAS  Article  PubMed  Google Scholar 

  37. Vembar, S.S., Jonikas, M.C., Hendershot, L.M., Weissman, J.S. & Brodsky, J.L. J domain co-chaperone specificity defines the role of BIP during protein translocation. J. Biol. Chem. 285, 22484–94 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Vanhove, M., Usherwood, Y.K. & Hendershot, L.M. Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release. Immunity 15, 105–114 (2001).

    CAS  Article  PubMed  Google Scholar 

  39. Knarr, G., Kies, U., Bell, S., Mayer, M. & Buchner, J. Interaction of the chaperone BiP with an antibody domain: implications for the chaperone cycle. J. Mol. Biol. 318, 611–620 (2002).

    CAS  Article  PubMed  Google Scholar 

  40. Wei, J., Gaut, J.R. & Hendershot, L.M. In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis. J. Biol. Chem. 270, 26677–26682 (1995).

    CAS  Article  PubMed  Google Scholar 

  41. Antonik, M., Felekyan, S., Gaiduk, A. & Seidel, C.A. Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. J. Phys. Chem. B 110, 6970–6978 (2006).

    CAS  Article  PubMed  Google Scholar 

  42. Kalinin, S., Felekyan, S., Antonik, M. & Seidel, C.A. Probability distribution analysis of single-molecule fluorescence anisotropy and resonance energy transfer. J. Phys. Chem. B 111, 10253–10262 (2007).

    CAS  Article  PubMed  Google Scholar 

  43. Wei, J. & Hendershot, L.M. Characterization of the nucleotide binding properties and ATPase activity of recombinant hamster BiP purified from bacteria. J. Biol. Chem. 270, 26670–26676 (1995).

    CAS  Article  PubMed  Google Scholar 

  44. Jiang, J., Prasad, K., Lafer, E.M. & Sousa, R. Structural basis of interdomain communication in the Hsc70 chaperone. Mol. Cell 20, 513–524 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Strub, A., Zufall, N. & Voos, W. The putative helical lid of the Hsp70 peptide-binding domain is required for efficient preprotein translocation into mitochondria. J. Mol. Biol. 334, 1087–1099 (2003).

    CAS  Article  PubMed  Google Scholar 

  46. Tokunaga, M., Kato, S., Kawamura-Watabe, A., Tanaka, R. & Tokunaga, H. Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein in Saccharomyces cerevisiae. Yeast 14, 1285–1295 (1998).

    CAS  Article  PubMed  Google Scholar 

  47. Schlecht, R., Erbse, A.H., Bukau, B. & Mayer, M.P . Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat. Struct. & Mol. Biol. (in the press).

  48. Shen, Y., Meunier, L. & Hendershot, L.M. Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress. J. Biol. Chem. 277, 15947–15956 (2002).

    CAS  Article  PubMed  Google Scholar 

  49. Vembar, S.S., Jin, Y., Brodsky, J.L. & Hendershot, L.M. The mammalian Hsp40 ERdj3 requires its Hsp70 interaction and substrate-binding properties to complement various yeast Hsp40-dependent functions. J. Biol. Chem. 284, 32462–32471 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Rodriguez, F. . et al. Molecular basis for regulation of the heat shock transcription factor sigma32 by the DnaK and DnaJ chaperones. Mol. Cell 32, 347–358 (2008).

    CAS  Article  PubMed  Google Scholar 

  51. Mayer, M.P., Rudiger, S. & Bukau, B. Molecular basis for interactions of the DnaK chaperone with substrates. Biol. Chem. 381, 877–885 (2000).

    CAS  Article  PubMed  Google Scholar 

  52. Liu, Q. & Hendrickson, W.A. Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1. Cell 131, 106–120 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Nørby, J.G. Coupled assay of Na+,K+-ATPase activity. Methods Enzymol. 156, 116–119 (1988).

    Article  PubMed  Google Scholar 

  54. Müller, B.K., Zaychikov, E., Brauchle, C. & Lamb, D.C. Pulsed interleaved excitation. Biophys. J. 89, 3508–3522 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Eggeling, C. et al. Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J. Biotechnol. 86, 163–180 (2001).

    CAS  Article  PubMed  Google Scholar 

  56. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Lee, N.K. et al. Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys. J. 88, 2939–2953 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Schäfer, H., Nau, K., Sickmann, A., Erdmann, R. & Meyer, H.E. Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry. Electrophoresis 22, 2955–2968 (2001).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank H. Krause for performing mass spectrometry experiments and V. Kudryavtsev for valuable discussions regarding analysis of the spFRET data. Funding of M.M. and M.J.F. by the Studienstiftung des deutschen Volkes, of M.H. by the International Doctorate Program NanoBioTechnology (IDK-NBT), of D.B. by the International Graduate School of Science and Engineering (IGSSE) and of J.B. by the SFB 749, the Fonds der chemischen Industrie and the Bayerische Forschungsstiftung is gratefully acknowledged. D.C.L. wishes to thank the Deutsche Forschungsgemeinschaft (SFB 749), the Center for Nano Science, the Ludwig-Maximilians-Universität Munich (LMUInnovativ BioImaging Network) and the Nanosystems Initative Munich (NIM) for financial support. The authors thank L. Hendershot for discussions and helpful comments on the manuscript.

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M.M., M.H., M.J.F., D.C.L. and J.B. designed the study and wrote the paper. M.M., M.J.F. and D.B. performed ensemble experiments and M.H. performed single molecule experiments. M.M., M.H., M.J.F. and D.C.L. analyzed data.

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Correspondence to Don C Lamb or Johannes Buchner.

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Marcinowski, M., Höller, M., Feige, M. et al. Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions. Nat Struct Mol Biol 18, 150–158 (2011). https://doi.org/10.1038/nsmb.1970

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