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Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53

Abstract

In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53.

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Figure 1: Interaction between Hsp90 and p53 fragments.
Figure 2: NMR analysis of the binding of the p53-DBD to the individual domains of Hsp90.
Figure 3: NMR analysis of the binding of single Hsp90 domains to the p53-DBD.
Figure 4: Structural model of the Hsp90-MD–p53-DBD complex.
Figure 5: Structural analysis of the Hsp90-CTD–p53-DBD interaction.
Figure 6: Chaperone activities of Hsp90 variants.

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References

  1. Ali, M.M. et al. Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006).

    Article  CAS  Google Scholar 

  2. Shiau, A.K., Harris, S.F., Southworth, D.R. & Agard, D.A. Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell 127, 329–340 (2006).

    Article  CAS  Google Scholar 

  3. Wandinger, S.K., Richter, K. & Buchner, J. The Hsp90 chaperone machinery. J. Biol. Chem. 283, 18473–18477 (2008).

    Article  CAS  Google Scholar 

  4. Wegele, H., Wandinger, S.K., Schmid, A.B., Reinstein, J. & Buchner, J. Substrate transfer from the chaperone Hsp70 to Hsp90. J. Mol. Biol. 356, 802–811 (2006).

    Article  CAS  Google Scholar 

  5. Fontana, J. et al. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ. Res. 90, 866–873 (2002).

    Article  CAS  Google Scholar 

  6. Hawle, P. et al. The middle domain of Hsp90 acts as a discriminator between different types of client proteins. Mol. Cell. Biol. 26, 8385–8395 (2006).

    Article  CAS  Google Scholar 

  7. Meyer, P. et al. Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol. Cell 11, 647–658 (2003).

    Article  CAS  Google Scholar 

  8. Nathan, D.F. & Lindquist, S. Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol. Cell. Biol. 15, 3917–3925 (1995).

    Article  CAS  Google Scholar 

  9. Harris, S.F., Shiau, A.K. & Agard, D.A. The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site. Structure 12, 1087–1097 (2004).

    Article  CAS  Google Scholar 

  10. Hessling, M., Richter, K. & Buchner, J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90. Nat. Struct. Mol. Biol. 16, 287–293 (2009).

    Article  CAS  Google Scholar 

  11. Park, S.J., Borin, B.N., Martinez-Yamout, M.A. & Dyson, H.J. The client protein p53 adopts a molten globule–like state in the presence of Hsp90. Nat. Struct. Mol. Biol. 18, 537–541 (2011).

    Article  CAS  Google Scholar 

  12. Rudiger, S., Freund, S.M., Veprintsev, D.B. & Fersht, A.R. CRINEPT-TROSY NMR reveals p53 core domain bound in an unfolded form to the chaperone Hsp90. Proc. Natl. Acad. Sci. USA 99, 11085–11090 (2002).

    Article  CAS  Google Scholar 

  13. Street, T.O., Lavery, L.A. & Agard, D.A. Substrate binding drives large-scale conformational changes in the hsp90 molecular chaperone. Mol. Cell 42, 96–105 (2011).

    Article  CAS  Google Scholar 

  14. Römer, L., Klein, C., Dehner, A., Kessler, H. & Buchner, J. p53–a natural cancer killer: structural insights and therapeutic concepts. Angew. Chem. Int. Edn Engl. 45, 6440–6460 (2006).

    Article  Google Scholar 

  15. Lane, D.P. & Crawford, L.V. T antigen is bound to a host protein in SV40-transformed cells. Nature 278, 261–263 (1979).

    Article  CAS  Google Scholar 

  16. Linzer, D.I. & Levine, A.J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43–52 (1979).

    Article  CAS  Google Scholar 

  17. Blagosklonny, M.V., Toretsky, J., Bohen, S. & Neckers, L. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc. Natl. Acad. Sci. USA 93, 8379–8383 (1996).

    Article  CAS  Google Scholar 

  18. Wang, C. & Chen, J. Phosphorylation and hsp90 binding mediate heat shock stabilization of p53. J. Biol. Chem. 278, 2066–2071 (2003).

    Article  CAS  Google Scholar 

  19. Whitesell, L. & Lindquist, S.L. HSP90 and the chaperoning of cancer. Nat. Rev. Cancer 5, 761–772 (2005).

    Article  CAS  Google Scholar 

  20. Müller, L., Schaupp, A., Walerych, D., Wegele, H. & Buchner, J. Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures. J. Biol. Chem. 279, 48846–48854 (2004).

    Article  Google Scholar 

  21. Sepehrnia, B., Paz, I.B., Dasgupta, G. & Momand, J. Heat shock protein 84 forms a complex with mutant p53 protein predominantly within a cytoplasmic compartment of the cell. J. Biol. Chem. 271, 15084–15090 (1996).

    Article  CAS  Google Scholar 

  22. Nagata, Y. et al. The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association. Oncogene 18, 6037–6049 (1999).

    Article  CAS  Google Scholar 

  23. Whitesell, L., Sutphin, P.D., Pulcini, E.J., Martinez, J.D. & Cook, P.H. The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent. Mol. Cell. Biol. 18, 1517–1524 (1998).

    Article  CAS  Google Scholar 

  24. Piper, P.W. et al. Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta, a property that can be exploited in screens for new Hsp90 chaperone inhibitors. Gene 302, 165–170 (2003).

    Article  CAS  Google Scholar 

  25. Richter, K. et al. Conserved conformational changes in the ATPase cycle of human Hsp90. J. Biol. Chem. 283, 17757–17765 (2008).

    Article  CAS  Google Scholar 

  26. Dehner, A. et al. Cooperative binding of p53 to DNA: regulation by protein-protein interactions through a double salt bridge. Angew. Chem. Int. Edn Engl. 44, 5247–5251 (2005).

    Article  CAS  Google Scholar 

  27. Dehner, A. et al. NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol. ChemBioChem 4, 870–877 (2003).

    Article  CAS  Google Scholar 

  28. Salek, R.M., Williams, M.A., Prodromou, C., Pearl, L.H. & Ladbury, J.E. Backbone resonance assignments of the 25kD N-terminal ATPase domain from the Hsp90 chaperone. J. Biomol. NMR 23, 327–328 (2002).

    Article  CAS  Google Scholar 

  29. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. USA 95, 13585–13590 (1998).

    Article  CAS  Google Scholar 

  30. Meyer, P. et al. Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. EMBO J. 23, 511–519 (2004).

    Article  CAS  Google Scholar 

  31. Retzlaff, M. et al. Asymmetric activation of the hsp90 dimer by its cochaperone aha1. Mol. Cell 37, 344–354 (2010).

    Article  CAS  Google Scholar 

  32. Sugase, K., Dyson, H.J. & Wright, P.E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).

    Article  CAS  Google Scholar 

  33. Wong, K.B. et al. Hot-spot mutants of p53 core domain evince characteristic local structural changes. Proc. Natl. Acad. Sci. USA 96, 8438–8442 (1999).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Cornilescu, G., Marquardt, J.L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998).

    Article  CAS  Google Scholar 

  36. Hagn, F. et al. BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain. J. Biol. Chem. 285, 3439–3450 (2010).

    Article  CAS  Google Scholar 

  37. Tomita, Y. et al. WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization. J. Biol. Chem. 281, 8600–8606 (2006).

    Article  CAS  Google Scholar 

  38. Friedler, A., Veprintsev, D.B., Rutherford, T., von Glos, K.I. & Fersht, A.R. Binding of Rad51 and other peptide sequences to a promiscuous, highly electrostatic binding site in p53. J. Biol. Chem. 280, 8051–8059 (2005).

    Article  CAS  Google Scholar 

  39. Wayne, N. & Bolon, D.N. Charge-rich regions modulate the anti-aggregation activity of Hsp90. J. Mol. Biol. 401, 931–939 (2010).

    Article  CAS  Google Scholar 

  40. Walerych, D. et al. ATP binding to Hsp90 is sufficient for effective chaperoning of p53 protein. J. Biol. Chem. 285, 32020–32028 (2010).

    Article  CAS  Google Scholar 

  41. Vaughan, C.K. et al. Structure of an Hsp90-Cdc37-Cdk4 complex. Mol. Cell 23, 697–707 (2006).

    Article  CAS  Google Scholar 

  42. Scheufler, C. et al. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101, 199–210 (2000).

    Article  CAS  Google Scholar 

  43. Fang, L., Ricketson, D., Getubig, L. & Darimont, B. Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain. Proc. Natl. Acad. Sci. USA 103, 18487–18492 (2006).

    Article  CAS  Google Scholar 

  44. Yamada, S., Ono, T., Mizuno, A. & Nemoto, T.K. A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone. FEBS J. 270, 146–154 (2003).

    CAS  Google Scholar 

  45. Friedler, A. et al. A peptide that binds and stabilizes p53 core domain: chaperone strategy for rescue of oncogenic mutants. Proc. Natl. Acad. Sci. USA 99, 937–942 (2002).

    Article  CAS  Google Scholar 

  46. Richter, K., Muschler, P., Hainzl, O., Reinstein, J. & Buchner, J. Sti1 is a non-competitive inhibitor of the Hsp90 ATPase. Binding prevents the N-terminal dimerization reaction during the ATPase cycle. J. Biol. Chem. 278, 10328–10333 (2003).

    Article  CAS  Google Scholar 

  47. Scheibel, T., Weikl, T. & Buchner, J. Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence. Proc. Natl. Acad. Sci. USA 95, 1495–1499 (1998).

    Article  CAS  Google Scholar 

  48. Ali, J.A., Jackson, A.P., Howells, A.J. & Maxwell, A. The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs. Biochemistry 32, 2717–2724 (1993).

    Article  CAS  Google Scholar 

  49. Schwarz, D. et al. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat. Protoc. 2, 2945–2957 (2007).

    Article  CAS  Google Scholar 

  50. Hayes, D.B. & Stafford, W.F. SEDVIEW, real-time sedimentation analysis. Macromol. Biosci. 10, 731–735 (2010).

    Article  CAS  Google Scholar 

  51. Demeler, B. UltraScan: a comprehensive data analysis software package for analytical ultracentrifugation experiments. in Modern Analytical Ultracentrifugation: Techniques and Methods (eds. D.J. Scott, Harding, S.E. & Rowe, A.J.) 210–229 (Royal Society of Chemistry (UK), 2005).

  52. Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).

    Article  CAS  Google Scholar 

  53. Jahnke, W., Bauer, C., Gemmecker, G. & Kessler, H. Improved accuracy of NMR structures by a modified NOESY-HSQC experiment. J. Magn. Reson. B. 106, 86–88 (1995).

    Article  CAS  Google Scholar 

  54. Leutner, M. et al. Automated backbone assignment of labeled proteins using the threshold accepting algorithm. J. Biomol. NMR 11, 31–43 (1998).

    Article  CAS  Google Scholar 

  55. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997).

    Article  CAS  Google Scholar 

  56. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997).

    Article  CAS  Google Scholar 

  57. Hansen, M.R., Mueller, L. & Pardi, A. Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat. Struct. Biol. 5, 1065–1074 (1998).

    Article  CAS  Google Scholar 

  58. Schwieters, C.D., Kuszewski, J.J., Tjandra, N. & Clore, G.M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003).

    Article  CAS  Google Scholar 

  59. de Vries, S.J., van Dijk, M. & Bonvin, A.M. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883–897 (2010).

    Article  CAS  Google Scholar 

  60. García de la Torre, J., Huertas, M.L. & Carrasco, B. HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations. J. Magn. Reson. 147, 138–146 (2000).

    Article  Google Scholar 

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft SFB594 and the Center for Integrated Protein Science Munich (J.B. and H.K.), the Elitenetzwerk Bayern (F.H. and J.R.) and the Fonds der chemischen Industrie (M.R.). F.H. is a recipient of an EMBO long-term fellowship (ALTF 265-2010). We want to thank D. Heckmann for peptide synthesis.

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F.H., S.L. and M.R. designed research, performed experiments, analyzed data and wrote the paper. J.R., O.D. and K.R. performed research and analyzed data. J.B. and H.K. designed research and wrote the paper. F.H. and S.L. carried out protein expression, interaction studies, resonance assignment, NMR interaction and docking studies. M.R. performed protein expression, interaction studies and activity assays.

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Correspondence to Johannes Buchner or Horst Kessler.

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Hagn, F., Lagleder, S., Retzlaff, M. et al. Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53. Nat Struct Mol Biol 18, 1086–1093 (2011). https://doi.org/10.1038/nsmb.2114

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