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Tuning protein autoinhibition by domain destabilization

Nature Structural & Molecular Biology volume 18, pages 550–555 (2011)Cite this article

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Abstract

Activation of many multidomain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. We established the molecular basis for this model for the archetypal signaling adaptor protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states. We used fluorescence and NMR spectroscopies together with segmental isotopic labeling by means of expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-like conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multidomain proteins.

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Figure 1: Scheme for the autoinhibition and activation of Crk-II.
Figure 2: NMR analysis of the cSH3 domain within full-length Crk-II.
Figure 3: Structural and dynamic analysis of the cSH3 domain within Crk-II.
Figure 4: Conformation of Trp275 modulates the stability of the cSH3 domain.

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References

  1. Schlessinger, J. Autoinhibition control. Science 300, 750–752 (2003).

    Article  CAS  Google Scholar 

  2. Ferguson, K.M. et al. EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol. Cell 11, 507–517 (2003).

    Article  CAS  Google Scholar 

  3. Smock, R.G. & Gierasch, L.M. Sending signals dynamically. Science 324, 198–203 (2009).

    Article  CAS  Google Scholar 

  4. Chen, L. et al. Structural insight into the autoinhibition mechanism of AMP-activated protein kinase. Nature 459, 1146–1149 (2009).

    Article  CAS  Google Scholar 

  5. Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452 (2003).

    Article  CAS  Google Scholar 

  6. Yao, X., Rosen, M.K. & Gardner, K.H. Estimation of the available free energy in a LOV2-Jα photoswitch. Nat. Chem. Biol. 4, 491–497 (2008).

    Article  CAS  Google Scholar 

  7. Li, P., Martins, I.R., Amarasinghe, G.K. & Rosen, M.K. Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nat. Struct. Mol. Biol. 15, 613–618 (2008).

    Article  CAS  Google Scholar 

  8. Miloushev, V.Z. et al. Dynamic properties of a type II cadherin adhesive domain: implications for the mechanism of strand-swapping of classical cadherins. Structure 16, 1195–1205 (2008).

    Article  CAS  Google Scholar 

  9. Muralidharan, V. et al. Domain-specific incorporation of noninvasive optical probes into recombinant proteins. J. Am. Chem. Soc. 126, 14004–14012 (2004).

    Article  CAS  Google Scholar 

  10. Tang, L., Roulhac, P.L. & Fitzgerald, M.C. H/D exchange and mass spectrometry-based method for biophysical analysis of multidomain proteins at the domain level. Anal. Chem. 79, 8728–8739 (2007).

    Article  CAS  Google Scholar 

  11. Feller, S.M. Crk family adaptors—signalling complex formation and biological roles. Oncogene 20, 6348–6371 (2001).

    Article  CAS  Google Scholar 

  12. Nojima, Y. et al. Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3–containing molecule having multiple Src homology 2-binding motifs. J. Biol. Chem. 270, 15398–15402 (1995).

    Article  CAS  Google Scholar 

  13. Schaller, M.D. & Parsons, J.T. pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol. Cell. Biol. 15, 2635–2645 (1995).

    Article  CAS  Google Scholar 

  14. Schaller, M.D. & Parsons, J.T. Focal adhesion kinase and associated proteins. Curr. Opin. Cell Biol. 6, 705–710 (1994).

    Article  CAS  Google Scholar 

  15. Knudsen, B.S., Feller, S.M. & Hanafusa, H. Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk. J. Biol. Chem. 269, 32781–32787 (1994).

    CAS  PubMed  Google Scholar 

  16. Ren, R., Ye, Z.S. & Baltimore, D. Abl protein-tyrosin kinase selects the Crk adapter as a substrate using SH3-binding sites. Genes Dev. 8, 783–795 (1994).

    Article  CAS  Google Scholar 

  17. Hasegawa, H. et al. DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane. Mol. Cell. Biol. 16, 1770–1776 (1996).

    Article  CAS  Google Scholar 

  18. Kiyokawa, E. et al. Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev. 12, 3331–3336 (1998).

    Article  CAS  Google Scholar 

  19. Kobashigawa, Y. et al. Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK. Nat. Struct. Mol. Biol. 14, 503–510 (2007).

    Article  CAS  Google Scholar 

  20. Feller, S.M., Knudsen, B. & Hanafusa, H. c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 13, 2341–2351 (1994).

    Article  CAS  Google Scholar 

  21. Sarkar, P., Reichman, C., Saleh, T., Birge, R.B. & Kalodimos, C.G. Proline cis-trans isomerization controls autoinhibition of a signaling protein. Mol. Cell 25, 413–426 (2007).

    Article  CAS  Google Scholar 

  22. Cowburn, D. Moving parts: how the adaptor protein CRK is regulated, and regulates. Nat. Struct. Mol. Biol. 14, 465–466 (2007).

    Article  CAS  Google Scholar 

  23. Mochizuki, N. et al. Crk activation of JNK via C3G and R-Ras. J. Biol. Chem. 275, 12667–12671 (2000).

    Article  CAS  Google Scholar 

  24. Wu, X. et al. Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. Structure 3, 215–226 (1995).

    Article  CAS  Google Scholar 

  25. Demers, J.-P. & Mittermaier, A. Binding mechanism of an SH3 domain studied by NMR and ITC. J. Am. Chem. Soc. 131, 4355–4367 (2009).

    Article  CAS  Google Scholar 

  26. Wildes, D. & Marqusee, S. Hydrogen exchange and ligand binding: ligand-dependent and ligand-independent protection in the Src SH3 domain. Protein Sci. 14, 81–88 (2005).

    Article  CAS  Google Scholar 

  27. Wang, C., Pawley, N.H. & Nicholson, L.K. The role of backbone motions in ligand binding to the c-Src SH3 domain. J. Mol. Biol. 313, 873–887 (2001).

    Article  CAS  Google Scholar 

  28. Cowburn, D. & Muir, T.W. Segmental isotopic labeling using expressed protein ligation. Methods Enzymol. 339, 41–54 (2001).

    Article  CAS  Google Scholar 

  29. Zvara, A. et al. Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-CrkII. Oncogene 20, 951–961 (2001).

    Article  CAS  Google Scholar 

  30. Lakowicz, J.R. Principles of Fluorescence Spectroscopy (Springer, New York, 2006).

  31. Eftink, M.R. & Ghiron, C.A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry 15, 672–680 (1976).

    Article  CAS  Google Scholar 

  32. Muralidharan, V. et al. Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II. Biochemistry 45, 8874–8884 (2006).

    Article  CAS  Google Scholar 

  33. Di Nardo, A.A., Larson, S.M. & Davidson, A.R. The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core. J. Mol. Biol. 333, 641–655 (2003).

    Article  CAS  Google Scholar 

  34. Zhou, H.X. Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains. Biophys. J. 91, 3170–3181 (2006).

    Article  CAS  Google Scholar 

  35. Donaldson, L.W., Gish, G., Pawson, T., Kay, L.E. & Forman-Kay, J.D. Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide. Proc. Natl. Acad. Sci. USA 99, 14053–14058 (2002).

    Article  CAS  Google Scholar 

  36. Porter, M., Schindler, T., Kuriyan, J. & Miller, W.T. Reciprocal regulation of Hck activity by phosphorylation of Tyr527 and Tyr416. J. Biol. Chem. 275, 2721–2726 (2000).

    Article  CAS  Google Scholar 

  37. Sarkar, P., Reichman, C., Saleh, T., Birge, R.B. & Kalodimos, C.G. Proline cis-trans isomerization controls autoinhibtion of a signaling protein. Mol. Cell 25, 413–426 (2007).

    Article  CAS  Google Scholar 

  38. 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 

  39. Blaschke, U.K., Cotton, G.J. & Muir, T.W. Synthesis of multi-domain proteins using expressed protein ligation: strategies for segmental isotopic labeling of internal regions. Tetrahedron 56, 9461–9470 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants GM070941 (D.P.R.), EB001991 (T.W.M.), GM55843 (T.W.M.) and GM59273 (A.G.P.). D.P.R., T.W.M. and A.G.P. are members of the New York Structural Biology Center (NYSBC) and are supported by NIH grant GM66354. We thank K. Dutta and S. Bhattacharya for their help in collecting NMR data at the NYSBC.

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  1. Vasant Muralidharan

    Present address: Present address: Howard Hughes Medical Institute, Washington University School of Medicine, Departments of Molecular Microbiology and Medicine, St. Louis, Missouri, USA.,

Authors and Affiliations

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

    Jae-Hyun Cho & Arthur G Palmer III

  2. The Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York, USA

    Vasant Muralidharan, Miquel Vila-Perello & Tom W Muir

  3. Department of Chemistry and Graduate Program in Biochemistry and Structural Biology, State University of New York at Stony Brook, Stony Brook, New York, USA

    Daniel P Raleigh

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Contributions

J.-H.C. designed and conducted all experiments, analyzed the data and helped write the paper. V.M. and M.V.-P. helped to synthesize the ligands and prepare the segmentally labeled protein. D.P.R., T.W.M. and A.G.P. designed the experiments, analyzed the data and helped write the paper.

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Correspondence to Tom W Muir or Arthur G Palmer III.

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Supplementary Figures 1–7 and Supplementary Methods (PDF 985 kb)

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Cho, JH., Muralidharan, V., Vila-Perello, M. et al. Tuning protein autoinhibition by domain destabilization. Nat Struct Mol Biol 18, 550–555 (2011). https://doi.org/10.1038/nsmb.2039

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