Prediction of the essential intermolecular contacts for side‐binding of VASP on F‐actin

Abstract

AbstractVasodilator‐stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly‐Pro region, a G‐actin‐binding domain (GAB), and an F‐actin‐binding domain (FAB). Together, the poly‐Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin–G‐actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly‐Pro region and GAB domain of VASP with profilin–actin, has been solved over a decade ago; however, a detailed structure of the FAB‐F‐actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F‐actin. Here, in order to develop our understanding of the VASP–actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain‐actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain‐level binding–unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side‐binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP–actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.