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Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

Nature Structural & Molecular Biology volume 20, pages 900–907 (2013)Cite this article

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Abstract

The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-Å resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.

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Figure 1: Characteristics of DnaK locked in an ATP-bound state.
Figure 2: Overall structure of DnaK-ATP.
Figure 3: Unique conformations of NBD and SBD in DnaK-ATP.
Figure 4: Two highly conserved glycine residues in L5,6.
Figure 5: Sequence conservation at domain interfaces in DnaK-ATP.
Figure 6: Characteristics of domain interfaces in DnaK-ATP.
Figure 7: Model for allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.

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References

  1. Hartl, F.U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    Article  CAS  Google Scholar 

  2. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    Article  CAS  Google Scholar 

  3. Bukau, B. & Horwich, A.L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366 (1998).

    Article  CAS  Google Scholar 

  4. Mayer, M.P. Gymnastics of molecular chaperones. Mol. Cell 39, 321–331 (2010).

    Article  CAS  Google Scholar 

  5. Zuiderweg, E.R. et al. Allostery in the Hsp70 Chaperone Proteins. Top. Curr. Chem. 328, 99–153 (2013).

    Article  CAS  Google Scholar 

  6. Rüdiger, S., Buchberger, A. & Bukau, B. Interaction of Hsp70 chaperones with substrates. Nat. Struct. Biol. 4, 342–349 (1997).

    Article  Google Scholar 

  7. Flaherty, K.M., DeLuca-Flaherty, C. & McKay, D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346, 623–628 (1990).

    Article  CAS  Google Scholar 

  8. Sriram, M., Osipiuk, J., Freeman, B., Morimoto, R. & Joachimiak, A. Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain. Structure 5, 403–414 (1997).

    Article  CAS  Google Scholar 

  9. Harrison, C.J., Hayer-Hartl, M., Di Liberto, M., Hartl, F. & Kuriyan, J. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276, 431–435 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Chang, Y.W., Sun, Y.J., Wang, C. & Hsiao, C.D. Crystal structures of the 70-kDa heat shock proteins in domain disjoining conformation. J. Biol. Chem. 283, 15502–15511 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Wang, H. et al. NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction. Biochemistry 37, 7929–7940 (1998).

    Article  CAS  Google Scholar 

  14. Pellecchia, M. et al. Structural insights into substrate binding by the molecular chaperone DnaK. Nat. Struct. Biol. 7, 298–303 (2000).

    Article  CAS  Google Scholar 

  15. Liebscher, M. & Roujeinikova, A. Allosteric coupling between the lid and interdomain linker in DnaK revealed by inhibitor binding studies. J. Bacteriol. 191, 1456–1462 (2009).

    Article  CAS  Google Scholar 

  16. Buchberger, A. et al. Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J. Biol. Chem. 270, 16903–16910 (1995).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Schmid, D., Baici, A., Gehring, H. & Christen, P. Kinetics of molecular chaperone action. Science 263, 971–973 (1994).

    Article  CAS  Google Scholar 

  20. Flynn, G.C., Chappell, T.G. & Rothman, J.E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245, 385–390 (1989).

    Article  CAS  Google Scholar 

  21. Flaherty, K.M., Wilbanks, S.M., DeLuca-Flaherty, C. & McKay, D.B. Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment. J. Biol. Chem. 269, 12899–12907 (1994).

    CAS  PubMed  Google Scholar 

  22. Jiang, J. et al. Structural basis of J cochaperone binding and regulation of Hsp70. Mol. Cell 28, 422–433 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Smock, R.G. et al. An interdomain sector mediating allostery in Hsp70 molecular chaperones. Mol. Syst. Biol. 6, 414 (2010).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Marcinowski, M. et al. Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions. Nat. Struct. Mol. Biol. 18, 150–158 (2011).

    Article  CAS  Google Scholar 

  27. Schlecht, R., Erbse, A.H., Bukau, B. & Mayer, M.P. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat. Struct. Mol. Biol. 18, 345–351 (2011).

    Article  CAS  Google Scholar 

  28. McCarty, J.S. & Walker, G.C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc. Natl. Acad. Sci. USA 88, 9513–9517 (1991).

    Article  CAS  Google Scholar 

  29. Osipiuk, J., Georgopoulos, C. & Zylicz, M. Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein. J. Biol. Chem. 268, 4821–4827 (1993).

    CAS  PubMed  Google Scholar 

  30. Xu, X. et al. The unique peptide substrate binding properties of 110 KDA heatshock protein (HSP110) determines its distinct chaperone activity. J. Biol. Chem. 287, 5661–5672 (2012).

    Article  CAS  Google Scholar 

  31. Smock, R.G., Blackburn, M.E. & Gierasch, L.M. Conserved, disordered C terminus of DnaK enhances cellular survival upon stress and DnaK in vitro chaperone activity. J. Biol. Chem. 286, 31821–31829 (2011).

    Article  CAS  Google Scholar 

  32. Liu, Q. et al. Structures from anomalous diffraction of native biological macromolecules. Science 336, 1033–1037 (2012).

    Article  CAS  Google Scholar 

  33. Swain, J.F. & Gierasch, L.M. The changing landscape of protein allostery. Curr. Opin. Struct. Biol. 16, 102–108 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Zhuravleva, A. & Gierasch, L.M. Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones. Proc. Natl. Acad. Sci. USA 108, 6987–6992 (2011).

    Article  CAS  Google Scholar 

  36. Kumar, D.P. et al. The four hydrophobic residues on the Hsp70 inter-domain linker have two distinct roles. J. Mol. Biol. 411, 1099–1113 (2011).

    Article  CAS  Google Scholar 

  37. Bhattacharya, A. et al. Allostery in Hsp70 chaperones is transduced by subdomain rotations. J. Mol. Biol. 388, 475–490 (2009).

    Article  CAS  Google Scholar 

  38. Rist, W., Graf, C., Bukau, B. & Mayer, M.P. Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation. J. Biol. Chem. 281, 16493–16501 (2006).

    Article  CAS  Google Scholar 

  39. Swain, J.F., Schulz, E.G. & Gierasch, L.M. Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states. J. Biol. Chem. 281, 1605–1611 (2006).

    Article  CAS  Google Scholar 

  40. Lovell, S.C. et al. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50, 437–450 (2003).

    Article  CAS  Google Scholar 

  41. Montgomery, D.L., Morimoto, R.I. & Gierasch, L.M. Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. J. Mol. Biol. 286, 915–932 (1999).

    Article  CAS  Google Scholar 

  42. Gässler, C.S. et al. Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone. Proc. Natl. Acad. Sci. USA 95, 15229–15234 (1998).

    Article  Google Scholar 

  43. Vogel, M., Bukau, B. & Mayer, M.P. Allosteric regulation of Hsp70 chaperones by a proline switch. Mol. Cell 21, 359–367 (2006).

    Article  CAS  Google Scholar 

  44. Davis, J.E., Voisine, C. & Craig, E.A. Intragenic suppressors of Hsp70 mutants: interplay between the ATPase- and peptide-binding domains. Proc. Natl. Acad. Sci. USA 96, 9269–9276 (1999).

    Article  CAS  Google Scholar 

  45. Polier, S., Dragovic, Z., Hartl, F.U. & Bracher, A. Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell 133, 1068–1079 (2008).

    Article  CAS  Google Scholar 

  46. Zhuravleva, A., Clerico, E.M. & Gierasch, L.M. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell 151, 1296–1307 (2012).

    Article  CAS  Google Scholar 

  47. Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863–874 (2012).

    Article  CAS  Google Scholar 

  48. Liu, Q., Liu, Q. & Hendrickson, W.A. Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data. Acta Crystallogr. D Biol. Crystallogr. (in the press).

  49. Mossessova, E. & Lima, C.D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    Article  CAS  Google Scholar 

  50. Burkholder, W.F. et al. Mutations in the C-terminal fragment of DnaK affecting peptide binding. Proc. Natl. Acad. Sci. USA 93, 10632–10637 (1996).

    Article  CAS  Google Scholar 

  51. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  52. Evans, P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).

    Article  CAS  Google Scholar 

  53. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

  54. Read, R.J. & McCoy, A.J. Using SAD data in Phaser. Acta Crystallogr. D Biol. Crystallogr. 67, 338–344 (2011).

    Article  CAS  Google Scholar 

  55. Cowtan, K.D. & Zhang, K.Y.J. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).

    Article  CAS  Google Scholar 

  56. Langer, G., Cohen, S.X., Lamzin, V.S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    Article  CAS  Google Scholar 

  57. Adams, P.D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).

    Article  CAS  Google Scholar 

  58. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  59. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  60. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  61. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  62. Becker, J., Walter, W., Yan, W. & Craig, E.A. Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo. Mol. Cell Biol. 16, 4378–4386 (1996).

    Article  CAS  Google Scholar 

  63. Lindahl, E., Hess, B. & van der Spoel, D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J. Mol. Model. 7, 306–317 (2001).

    Article  CAS  Google Scholar 

  64. Balsera, M.A., Wriggers, W., Oono, Y. & Schulten, K. Principal component analysis and long time protein dynamics. J. Phys. Chem. 100, 2567–2572 (1996).

    Article  CAS  Google Scholar 

  65. Ichiye, T. & Karplus, M. Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations. Proteins 11, 205–217 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank E. Craig, D. Logothetis, G. Tseng, L. Avery, L. Greene, J. Rife and C. Fox for critically reading the manuscript and providing insightful suggestions. We are grateful to J. Schwanof, R. Abramowitz and X. Yang (Brookhaven National Laboratory Beamline X4A and X4C) for their assistance in collecting diffraction data. We thank D. Kumar for technical support and C. Escalante for the PC1 photon counting spectrofluorimeter. This work was supported by startup funds from the Virginia Commonwealth University School of Medicine (to Qinglian Liu), a New Scholar Award in Aging from the Ellison Medical Foundation (AG-NS-0587-09 to Qinglian Liu) and a Grant-In-Aid Award from the American Heart Association (11GRNT7460003 to Qinglian Liu).

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  1. Ruifeng Qi, Evans Boateng Sarbeng, Qun Liu and Katherine Quynh Le: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA

    Ruifeng Qi, Evans Boateng Sarbeng, Katherine Quynh Le, Xinping Xu, Hongya Xu, Jiao Yang, Jennifer Li Wong, Christina Vorvis, Lei Zhou & Qinglian Liu

  2. New York Structural Biology Center, X4 Beamlines, Brookhaven National Laboratory, Upton, New York, USA

    Qun Liu & Wayne A Hendrickson

  3. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Wayne A Hendrickson

  4. Department of Physiology and Cellular Biophysics, Columbia University, New York, New York, USA

    Wayne A Hendrickson

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Contributions

Qinglian Liu designed the study and experiments. Qinglian Liu and L.Z. screened, identified and carried out the cloning for the crystallization construct of full-length DnaK. R.Q. and E.B.S. prepared the crystals for full-length DnaK and isolated SBD, respectively. Qun Liu and Qinglian Liu performed the structure determination and modeling. R.Q., E.B.S., K.Q.L., X.X., H.X., J.Y., J.L.W. and C.V. carried out the cloning of the mutations for biochemical analysis, protein purification, biochemical and genetic analysis. L.Z. performed computational analysis on the structures. Qinglian Liu, W.A.H. and L.Z. analyzed the structures, carried out data analysis and wrote the manuscript.

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Correspondence to Qinglian Liu.

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Qi, R., Sarbeng, E., Liu, Q. et al. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat Struct Mol Biol 20, 900–907 (2013). https://doi.org/10.1038/nsmb.2583

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