Article | Published:

Tuning protein autoinhibition by domain destabilization

Nature Structural & Molecular Biology volume 18, pages 550555 (2011) | Download Citation

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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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.

Author information

Author notes

    • Vasant Muralidharan

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

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Tom W Muir or Arthur G Palmer III.

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DOI

https://doi.org/10.1038/nsmb.2039

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