Biochemical and Biophysical Characterization of the Allosteric Equilibrium of the Wiskott-Aldrich Syndrome Protein
Leung, Daisy W.
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Proteins provide the essential building blocks of signal transduction pathways governing many biological processes. They are dynamic entities endowed with properties that allow them to respond to changes in the cell while maintaining specificity and fidelity of signaling in a crowded intracellular environment. Many signaling proteins are regulated by a subset of allostery where intramolecular interactions modulate the conformational equilibrium between an uninhibited, inactive state and an active state. Relief of autoinhibition then requires that covalent modification or binding events shift the equilibrium to favor the active state. However, the structural, biochemical, and biophysical properties of many autoinhibited systems have not been characterized. Thus, an understanding of how binding events are coupled to effector activation remains incomplete. Previous work in our lab has described a framework based upon classical descriptions of allostery with which we can examine the regulation and activation of the Wiskott-Aldrich Syndrome protein (WASP) by the Rho-family GTPase Cdc42. Results from this work revealed that a simple two-state model can predict the hydrogen exchange behavior and binding affinity for Cdc42-GTP of WASP proteins over a range of folding stabilities. The goals of my thesis have been: 1) to further develop the quantitative twostate model of the allosteric regulation of WASP, 2) to understand how binding of Cdc42 is coupled to WASP activation toward Arp2/3 complex-mediated actin polymerization, and 3) to understand the implications of the GTPase nucleotide switch on effector activation. In order to expand upon the range of WASP protein folding stabilities and to, more importantly, examine the relationship between Cdc42 binding and WASP activity towards the Arp2/3 actin nucleation complex, I generated a different series of WASP proteins by introducing mutations in the autoinhibited core of a more physiologically relevant WASP construct, which I used to parameterize our two-state model. Model predictions of WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 are all borne out experimentally and are functions of the two-state allosteric equilibrium of WASP. Application of the model to Cdc42-GDP revealed that the ratio of binding affinities for the inactive and active states of WASP is significantly smaller for Cdc42-GTP than for Cdc42-GDP. Thus, the GTP-bound state of Cdc42 is more effective at distinguishing between the two states of WASP, converting Cdc42-GDP from a partial agonist to a full agonist of WASP. Therefore, the nucleotide switch in Cdc42 is not only based upon a change in affinity for the two states of WASP, but also the efficiency of coupling between the binding and allosteric equilibria of WASP. These properties have important implications for how specificity and fidelity of signaling can be maintained in a crowded intracellular environment, ensuring that only Cdc42-GTP can activate WASP and signal downstream.