Dynamic Origins of Interdomain Cooperativity in the Vav1 Proto-Oncoprotein

Multi-domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains. In many such proteins a core regulatory interaction, involving binding of an inhibitory domain to the active site of a catalytic domain, is cooperatively enhanced by additional intramolecular domain-domain contacts. However, the physical basis of this cooperativity, and thus the energetic construction of multi-domain systems, is not well understood. The five-domain N-terminal regulatory element of the Vav1 proto-oncoprotein is representative of this class of signaling molecules. The catalytic activity of the Vav1 Dbl homology (DH) domain is autoinhibited by binding of an adjacent helix into the enzyme active site. This core inhibitory process is cooperatively enhanced through an unknown mechanism by intramolecular contacts of the N-terminal calponin homology (CH) domain of the protein. Here we show by NMR spectroscopy that the isolated helix-DH module 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. The rate of phosphorylation of the helix, an event that relieves autoinhibition, is linearly related to the population of the excited state. Thus, phosphorylation only occurs efficiently through the excited state, and internal dynamics are required for and control the rate of activation of the helix-DH module. In the full five-domain element this regulatory equilibrium is further biased 10-20-fold toward the closed state, implying that regulatory cooperativity derives from thermodynamic coupling between the helix-DH equilibrium and other interdomain binding equilibria in the protein. This explains the transforming activity of truncated Vav1 proteins lacking the CH domain, and suggests that Vav1 activation in vivo likely involves integration of kinase signals and signals to the CH or other domains of the molecule.