Characterizaton of Vinculin tail-PIP2 Structure and Binding (2014)

Undergraduates: Mihir Pershad, Peter Thompson

Faculty Advisor: Sharon Campbell
Department: Chemistry

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Vinculin is an essential cytoskeletal protein present in focal adhesions and cell-cell adhesions that plays an important role in cell adhesion, motility, and force transmission, by binding to multiple ligands. The interaction between the tail domain of vinculin (Vt) and the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP¿2) at the cell membrane is thought to have an important role in PIP2 signaling, a pathway significant in cell movement. This study sought to determine the specific residues involved in PIP2 binding by lipid co-sedimentation assays and to develop a PIP¿2-bound vinculin structure by x-ray crystallography. Three residues in Vt were identified as having a critical role in PIP2 binding. Vinculin variants containing mutations of these residues were tested in lipid binding and co-sedimentation assays with PIP2-containing liposomes and were found to significantly disrupt binding and membrane insertion of vinculin. To date, x-ray crystallography of these variants has not yielded a conclusive structure. Optimal conditions for crystal growth are currently being studied to develop larger crystals that will yield brighter x-ray diffraction patterns and a conclusive structure.