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Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7

A Corrigendum to this article was published on 15 February 2013

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Abstract

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

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Figure 1: Identification of two families of ubiquitin variants with high affinity for USP7 (U7Ubs).
Figure 2: Affinity maturation of the surface of a non-disulfide U7Ub further improves affinity for USP7.
Figure 3: Crystal structures of U7Ubs reveal altered backbone conformations or core packing.
Figure 4: Both core-repacking and solvent-exposed mutations are necessary for U7Ub25 to achieve high affinity.
Figure 5: U7Ub25 and U7Ub25.2540 are potent and specific inhibitors of full-length USP7.
Figure 6: U7Ub25.2540 variants are selective inhibitors of endogenous USP7 in the cellular environment.

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  • 29 January 2013

    In the version of this article initially published, the authors neglected to acknowledge an important collaborator and to include a citation of their related work. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Rizk, S.S. et al. Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Nat. Struct. Mol. Biol. (2011).

  2. Gao, J., Sidhu, S.S. & Wells, J.A. Two-state selection of conformation-specific antibodies. Proc. Natl. Acad. Sci. USA 106, 3071–3076 (2009).

    Article  CAS  Google Scholar 

  3. Lange, O.F. et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 (2008).

    Article  CAS  Google Scholar 

  4. Clague, M.J. & Urbé, S. Ubiquitin: same molecule, different degradation pathways. Cell 143, 682–685 (2010).

    Article  CAS  Google Scholar 

  5. Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    Article  CAS  Google Scholar 

  6. Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).

    Article  CAS  Google Scholar 

  7. Humphris, E.L. & Kortemme, T. Design of multi-specificity in protein interfaces. PLoS Comput. Biol. 3, e164 (2007).

    Article  Google Scholar 

  8. Friedland, G.D., Lakomek, N.-A., Griesinger, C., Meiler, J. & Kortemme, T. A correspondence between solution-state dynamics of an individual protein and the sequence and conformational diversity of its family. PLoS Comput. Biol. 5, e1000393 (2009).

    Article  Google Scholar 

  9. Komander, D., Clague, M.J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

    Article  CAS  Google Scholar 

  10. Nicholson, B. & Suresh Kumar, K.G. The multifaceted roles of USP7: new therapeutic opportunities. Cell Biochem. Biophys. 60, 61–68 (2011).

    Article  CAS  Google Scholar 

  11. Hussain, S., Zhang, Y. & Galardy, P.J. DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors. Cell Cycle 8, 1688–1697 (2009).

    Article  CAS  Google Scholar 

  12. Li, M., Brooks, C.L., Kon, N. & Gu, W. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 13, 879–886 (2004).

    Article  CAS  Google Scholar 

  13. Li, M. et al. Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648–653 (2002).

    Article  CAS  Google Scholar 

  14. Faesen, A.C. et al. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase. Mol. Cell 44, 147–159 (2011).

    Article  CAS  Google Scholar 

  15. Fernández-Montalván, A. et al. Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization. FEBS J. 274, 4256–4270 (2007).

    Article  Google Scholar 

  16. Ernst, A. Science 10.1126/science.1230161 (3 January 2013).

  17. Lowman, H.B. & Wells, J.A. Affinity maturation of human growth hormone by monovalent phage display. J. Mol. Biol. 234, 564–578 (1993).

    Article  CAS  Google Scholar 

  18. Levin, A.M. et al. Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'. Nature 484, 529–533 (2012).

    Article  CAS  Google Scholar 

  19. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

    Article  CAS  Google Scholar 

  20. Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).

    Article  CAS  Google Scholar 

  21. Yuan, J., Luo, K., Zhang, L., Cheville, J.C. & Lou, Z. USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 140, 384–396 (2010).

    Article  CAS  Google Scholar 

  22. Huang, O.W. et al. Phosphorylation-dependent activity of the deubiquitinase DUBA. Nat. Struct. Mol. Biol. 19, 171–175 (2012).

    Article  CAS  Google Scholar 

  23. Reyes-Turcu, F.E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).

    Article  CAS  Google Scholar 

  24. Renatus, M. et al. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure 14, 1293–1302 (2006).

    Article  CAS  Google Scholar 

  25. Ganesan, R. et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody. Structure 17, 1614–1624 (2009).

    Article  CAS  Google Scholar 

  26. Ultsch, M.H., Somers, W., Kossiakoff, A.A. & de Vos, A.M. The crystal structure of affinity-matured human growth hormone at 2-Å resolution. J. Mol. Biol. 236, 286–299 (1994).

    Article  CAS  Google Scholar 

  27. Lazar, G.A., Johnson, E.C., Desjarlais, J.R. & Handel, T.M. Rotamer strain as a determinant of protein structural specificity. Protein Sci. 8, 2598–2610 (1999).

    Article  CAS  Google Scholar 

  28. The Practice of Medicinal Chemistry, 2nd edn. (ed. Wermuth, C.G.) (Academic Press, 2003).

  29. Giebel, L.B. et al. Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities. Biochemistry 34, 15430–15435 (1995).

    Article  CAS  Google Scholar 

  30. O'Neil, K.T. et al. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins 14, 509–515 (1992).

    Article  CAS  Google Scholar 

  31. Tzeng, S.-R. & Kalodimos, C.G. Dynamic activation of an allosteric regulatory protein. Nature 462, 368–372 (2009).

    Article  CAS  Google Scholar 

  32. Tzeng, S.-R. & Kalodimos, C.G. Protein activity regulation by conformational entropy. Nature 488, 236–240 (2012).

    Article  CAS  Google Scholar 

  33. Hammes, G.G., Benkovic, S.J. & Hammes-Schiffer, S. Flexibility, diversity, and cooperativity: pillars of enzyme catalysis. Biochemistry 50, 10422–10430 (2011).

    Article  CAS  Google Scholar 

  34. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

    Article  CAS  Google Scholar 

  35. Dong, K.C. et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011).

    Article  CAS  Google Scholar 

  36. Tonikian, R., Zhang, Y., Boone, C. & Sidhu, S.S. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat. Protoc. 2, 1368–1386 (2007).

    Article  CAS  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  38. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  39. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  Google Scholar 

  40. Lange, O.F., van der Spoel, D. & de Groot, B.L. Scrutinizing molecular mechanics force fields on the submicrosecond timescale with NMR data. Biophys. J. 99, 647–655 (2010).

    Article  CAS  Google Scholar 

  41. Cai, M. et al. An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J. Biomol. NMR 11, 97–102 (1998).

    Article  CAS  Google Scholar 

  42. Hansen, D.F. et al. An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange. J. Am. Chem. Soc. 129, 11468–11479 (2007).

    Article  CAS  Google Scholar 

  43. Bahrami, A., Assadi, A.H., Markley, J.L. & Eghbalnia, H.R. Probabilistic interaction network of evidence algorithm and its application to complete labeling of peak lists from protein NMR spectroscopy. PLoS Comput. Biol. 5, e1000307 (2009).

    Article  Google Scholar 

  44. Findeisen, M., Brand, T. & Berger, S. A 1H-NMR thermometer suitable for cryoprobes. Magn. Reson. Chem. 45, 175–178 (2007).

    Article  CAS  Google Scholar 

  45. Grzesiek, S. & Bax, A. The importance of not saturating water in protein NMR. Application to sensitivity enhancement and NOE measurements. J. Am. Chem. Soc. 115, 12593–12594 (1993).

    Article  CAS  Google Scholar 

  46. Hansen, D.F., Feng, H., Zhou, Z., Bai, Y. & Kay, L.E. Selective characterization of microsecond motions in proteins by NMR relaxation. J. Am. Chem. Soc. 131, 16257–16265 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, J.D. & Kay, L.E. Slow dynamics in folded and unfolded states of an SH3 domain. J. Am. Chem. Soc. 123, 11341–11352 (2001).

    Article  CAS  Google Scholar 

  49. Mulder, F.A.A., Skrynnikov, N.R., Hon, B., Dahlquist, F.W. & Kay, L.E. Measurement of slow (μs-ms) time scale dynamics in protein side chains by 15N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J. Am. Chem. Soc. 123, 967–975 (2001).

    Article  CAS  Google Scholar 

  50. Wertz, I.E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).

    Article  CAS  Google Scholar 

  51. Newton, K. et al. Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation. Methods Mol. Biol. 832, 185–196 (2012).

    Article  CAS  Google Scholar 

  52. Matsumoto, M.L. et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 10.1016/j.jmb.2011.12.053 (2011).

  53. Matsumoto, M.L. et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol. Cell 39, 477–484 (2010).

    Article  CAS  Google Scholar 

  54. Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the DNA synthesis and sequencing groups for invaluable assistance in creating and isolating variants. We also thank E. Dueber, B. Lazarus and W. Fairbrother for critical reading of the manuscript and I. Chen for assistance in purifying U7Ub7.

We apologize for inadvertently omitting the acknowledgment of the following important collaborators. We thank S. Sidhu and A. Ernst for advice on ubiquitin surface library design and USP7 protein production. We also thank M. Kwok, Y. Franke and K. Bowman for the USP7cd* construct. A manuscript describing the use of surface-engineered ubiquitin variants to inhibit ubiquitin-signaling enzymes has recently been published, and we have added a citation to this work.

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Y.Z. and L.Z. performed mutagenesis, protein expression and purification, phage display, phage ELISA, protein ELISA, biolayer interferometry and enzymatic assays. L.R. and J.M.M. crystallized proteins and solved structures. A.H.P. performed NMR analysis. P.L. and W.S. performed MS. E.H. expressed and purified proteins. I.E.W. and C.L. performed the cellular experiments. J.E.C. performed computational design and modeling and measured affinities by ITC. All authors designed, performed and analyzed experiments. All authors contributed to writing the paper, with Y.Z. and J.E.C. coordinating.

Corresponding author

Correspondence to Jacob E Corn.

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All authors are employees of Genentech.

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Zhang, Y., Zhou, L., Rouge, L. et al. Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7. Nat Chem Biol 9, 51–58 (2013). https://doi.org/10.1038/nchembio.1134

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