Novel Bivalent Interaction between VASP-EVH1 And Zyxin is Critical for Binding Orientation

Abstract

The interaction of EVH1 domain of the actin polymerization regulatory protein VASP with the cytoskeletal protein Zyxin shows a novel secondary binding site by NMR spectroscopy. Previous studies had shown the importance of the Y39 phosphorylation in EVH1 for co-localization of VASP and Zyxin in the cell. We found a novel secondary binding site that is located in the structural vicinity of Y39. Phosphomimetic mutation Y39E eliminates binding to this secondary interaction site. By analyzing NMR populations, we determined that the secondary binding site provides an orientation preference for the four-motif Zyxin ligand. We further ran double/triple resonance NMR experiments, including carbon-detected, to determine the resonance assignments and structure of a chimera that includes EVH1 and motifs two-four of Zyxin. This structure shows EVH1 bound simultaneously and specifically to two of the four proline-rich binding motifs in Zyxin. This works reveals a secondary binding site in EVH1 that could represent a previously unrecognized coordination mechanism for partners with multiple proline-rich binding motifs.