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Internal dynamics control activation and activity of the autoinhibited Vav DH domain

Abstract

Protein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here we show by NMR spectroscopy that the autoinhibited DH domain exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain.

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Figure 1: Relaxation dispersion in AD and structural mapping of dynamic residues.
Figure 2: AD mutants differentially sample the helix-DH equilibrium.
Figure 3: Conformational equilibrium of the inhibitory helix controls the rate of AD phosphorylation.
Figure 4: Conformational equilibrium of the inhibitory helix controls GEF activity.
Figure 5: Model for function and regulation of the AD module of Vav1.

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Acknowledgements

We thank C. Amezcua for technical assistance with NMR data acquisition, L.E. Kay for providing the pulse sequences used in this work, L.E. Kay and D.M. Korzhnev for assistance with data analysis and J. Umetani for continuous support with insect cell culture. G.K.A. was supported by a postdoctoral fellowship from the Cancer Research Institute (USA). I.R.S.M. was supported by a Ph.D. fellowship from the Fundação para a Ciência e a Tecnologia (Portugal). This work was supported by US National Institutes of Health grant GM066930 to M.K.R.

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Contributions

M.K.R. conceived of the project; G.K.A. managed the work; P.L. and I.R.S.M. recognized the four-state problem; P.L. conceived of the mutagenesis strategy to solve the four-state problem; P.L. and I.R.S.M. generated the AD mutant series; P.L. and I.R.S.M. performed NMR studies of the AD mutants; and G.K.A. developed methods to generate pAD proteins and performed phosphorylation assays on the AD mutants. P.L. and I.R.S.M. performed GEF assays in the AD mutants; M.K.R., P.L., I.R.S.M. and G.K.A. analyzed the data and wrote the paper.

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Correspondence to Michael K Rosen.

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Li, P., Martins, I., Amarasinghe, G. et al. Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nat Struct Mol Biol 15, 613–618 (2008). https://doi.org/10.1038/nsmb.1428

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