Together the NMR and biochemical data support the model for function and regulation of the AD element of Vav1 shown in . The AD protein exists in an equilibrium between a ground state conformation where the inhibitory helix is bound to the DH domain as observed in the solution structure, and an excited state resembling pAD, where the helix is dissociated from the DH domain and melted. In the ground state, Tyr174 is buried in the interface of the inhibitory helix with the active site of the DH domain. These contacts block access of kinases to Tyr174 and of GTPase substrates to the DH active site. In the excited state, Tyr174 and the DH active site are accessible, and phosphorylation rate and GEF catalysis are those of the free Tyr174 site and free DH domain, respectively. A second equilibrium within the DH domain core (not shown in ) is coupled to the helix dynamics; work is underway to determine the functional significance, if any, of this additional process. Under the conditions used in the assays here, the conformational transitions of the helix are rapid relative to phosphorylation and GEF catalysis, so for any given protein the measured rates of these activities are proportional to the population of the open state. In wild type AD, the equilibrium is strongly biased toward the helix-bound state (~90 %, ), and phosphorylation rate and GEF activity are low. Upon phosphorylation, the protein is driven strongly toward the helix-dissociated/melted state where it is fully active. Thus, internal dynamics of the autoinhibited AD module play a central role in dictating its basal catalytic activity and in enabling its full activation by tyrosine kinases.
Model for function and regulation of the AD module of Vav1. See text for explanation.
Characterization of the open population of the autoinhibited AD protein provides an explanation for a puzzling observation in the literature: the Y174F mutant of full length Vav1 has appreciable transforming activity in vivo
, even though it cannot undergo the activating phosphorylation event (and thus should be constitutively inactive)6
. Our NMR analysis revealed that ADY174F
is 99 % open, versus 9 % for the wild type protein (). This change corresponds to c.a. 4.2 kcal mol−1
decrease in the free energy of the open/active state relative to the closed/inactive state. Concomitantly, the Y174F mutation increases the relative GEF activity by about 11-fold in AD, and a similar effect was seen with the full-length regulatory element (). Thus our data explain the enhanced activation of Rac and cell transformation induced by this mutation.
A recent EM reconstruction of Vav3 has suggested that the CH domain interacts with the ZF domain and perhaps the Ac region20
. These interactions appear to act on the AD element to cooperatively suppress the DH domain, since mutations to Vav that incapacitate or eliminate the CH domain lead to increased GEF activity and resultant cell transformation20-23
. Our characterization of the AD element explains the effects of CH perturbations in quantitative terms. In such mutants, even though the core autoinhibitory module (AD) is intact, the inherent thermodynamics of the module produce substantial populations of the active state and probably higher steady state phosphorylation level as well. When such truncated proteins are overexpressed, this population could easily be sufficient to cause transformation. This construction of Vav, with a core active site repression mechanism that is modulated by contacts of other modular elements, is often observed in autoinhibited multi-domain systems24,25
. It is clear in many cases that the modulatory interactions both suppress activity in the basal state, and provide mechanisms of integrating multiple inputs to achieve signaling specificity in vivo4,24
. However the energetic bases of this modulation have generally not been explored, and could involve changes to the kinetics, thermodynamics and/or structure of the core and its interconverting states. Our characterization of the AD core of Vav1 now sets the stage to understand how the physical properties of this element are modulated by interactions of other domains in the protein to provide the level of suppression and control of DH activity necessary for in vivo