The role of pH, self-association, and lipid binding in vinculin tail structure and function Public Deposited

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  • March 22, 2019
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  • Palmer, Sean Matthew
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Vinculin is a highly conserved cytoskeletal protein that localized to sites of cell adhesion and is involved in linking the actin cytoskeleton to the cell membrane. Loss of normal vinculin function has been associated with cancer phenotypes, cardiovascular disease, and lethal errors in embryogenesis. Vinculin is composed of a 90 kDa head domain (Vh) and a 21 kDa tail domain (Vt) connected by a flexible "neck" region. Vh and Vt form auto-inhibitory interactions in its inactive state. Conformational changes in these domains have been proposed to be important for vinculin activation, freeing domains to bind to a variety of ligands involved in cell adhesion processes. My work has focused on the vinculin tail domain. I have assigned the majority of the backbone 1HN, 15N, 13C[alpha], 13CO, and side chain 13C[beta] NMR resonances of Vt (Biological Magnetic Resonance Data Bank, accession number 15653). These assignments have proven useful for investigating the effects of ligand binding, pH and Vt self-association. The tail domain of vinculin has been reported to undergo a pH dependent conformational change between pH 5.5 and 7.5. However, we found no indication of a significant alteration in Vt conformation in this pH range by both circular dichroism and NMR. Although, Vt was reported to self-associate at pH 5.5 while remaining monomeric at pH 7.0, results from analytical ultracentrifugation indicate that Vt self-associates at both pH values, albeit with weak affinity (Kd > 100 [mu]M). The binding of acidic phospholipids by Vt has been proposed to play a role in vinculin activation and focal adhesion turnover. We observe a marked affinity of Vt for phosphatidylinositol 4,5-bisphosphate (PIP2), but no significant binding to phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol (PI) in the context of mixed lipid vesicles. A significant increase in PIP2 binding was observed with multiple mutant forms of Vt, expected to alter the conformation or flexibility of the N-terminal strap of Vt, suggesting that a rearrangement of this N-terminal strap may be required for PIP2 binding. Additionally, we find that the hydrophobic hairpin at the C-terminus of Vt is not critical for lipid insertion, as previously proposed.
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  • Campbell, Sharon
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