PIP2-induced membrane binding of the vinculin tail competes with its other binding partners.

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, in which unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond-long all-atom simulations. This allowed us to map the interaction of the vinculin tail with PIP2-enriched membranes in atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the basic collar and the basic ladder, as the main PIP2 interaction sites. By introducing a valency-disaggregated binding network analysis, we were able to map the protein-lipid interactions in unprecedented detail. In contrast to the basic collar, in which PIP2 is specifically recognized by an up to hexavalent binding pocket, the basic ladder forms a series of low-valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining vinculin in a closed, autoinhibited conformation. These findings led us to propose a molecular mechanism for the coupling between vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-actin binding that disfavors simultaneous interaction with both ligands, despite nonoverlapping binding sites.