Characterization of Internal Dynamics in Vav 1: Method Development, Mutual Coupling and Functional

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Title: Characterization of Internal Dynamics in Vav 1: Method Development, Mutual Coupling and Functional
Author: Li, Pilong
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 I show by NMR spectroscopy that the autoinhibited DH domain (AD ) 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 . Vav1 belong to a class of multi -domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains . In many such proteins there is a core regulatory interaction , involving binding of an inhibitory element to the active site of a functional domain like the inhibitory helix to DH in Vav1 . The core interaction is cooperatively enhanced by additional intramolecular domain -domain contacts . The physical basis of this cooperativity , and thus the energetic construction of multi -domain systems , is not well understood . Dynamics analysis of AD reveals that the closed and open populations are about 10 :1 for the core interaction in isolation . In the full five -domain regulatory fragment of Vav1 , interactions between domains outside of the core further bias this inhibitory equilibrium ~10 -fold toward the closed state , further suppressing activity . Thus , Vav1 is controlled by two , weakly biasing , but thermodynamically coupled equilibria - -an energetic construction that is probably general among multi -domain proteins . The dynamic landscape of AD is composed of two ?s -ms time scale motions : one is the inhibitory helix binding to and dissociating from the DH domain and another is intrinsic to the DH domain . Interestingly relative populations and exchange rates of the second process are altered upon perturbations to the inhibitory helix , suggesting that the two dynamic processes are energetically and kinetically coupled . A strategy has been established to quantify the thermodynamic and kinetic coupling strengths between the two processes via direction parameterization of four -state equilibria using NMR Carr -Purcell -Meiboom -Gill measurement . The coupling strengths between the two dynamic processes in AD are 1 .0 ~1 .5 kcal M -1 comparable to the coupling strength between the modulatory interaction and the helix -DH interactions in the full five -domain regulatory fragment of Vav1 . The coupling strength is relatively weak consistent with the coupling strengths reported for many other signaling proteins such as Src tyrosine kinase . These findings suggest that weakly coupling may be a common theme in regulatory molecules .
URI: http : / /hdl .handle .net /2152 .5 /689
Date: 2009-09-04


Characterization of Internal Dynamics in Vav 1: Method Development, Mutual Coupling and Functional. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /689 .

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