Lipid Binding to the Tail Domain of Vinculin

Sean M Palmer,Martin P Playford,Susan W Craig,Michael D Schaller,Sharon L Campbell

doi:10.1074/jbc.m807842200

Abstract

Vinculin is a highly conserved and abundant cytoskeletal protein involved in linking the actin cytoskeleton to the cell membrane at sites of cellular adhesion. At these sites of adhesion, vinculin plays a role in physiological processes such as cell motility, migration, development, and wound healing. Loss of normal vinculin function has been associated with cancer phenotypes, cardiovascular disease, and lethal errors in embryogenesis. The tail domain of vinculin (Vt) binds to acidic phospholipids and has been proposed to play a role in vinculin activation and focal adhesion turnover. To better characterize Vt-lipid specificity, we conducted a series of lipid co-sedimentation experiments and find that Vt shows specific association with phosphatidylinositol 4,5-bisphosphate (PIP2), compared with phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol (PI) in the context of mixed lipid vesicles. The C terminus of Vt has been proposed to be important for PIP2 association, as various mutations and deletions within the C-terminal reduce PIP2 association. Lipid co-sedimentation and NMR analyses indicate that removal of the hydrophobic hairpin does not alter Vt structure or PIP2 association. However, more extensive deletions within the C-terminal introduce Vt structural perturbations and reduce PIP2 binding. Intriguingly, a significant increase in PIP2 binding was observed for multiple Vt variants that perturb interactions between the N-terminal strap and helix bundle, suggesting that a rearrangement of this N-terminal strap may be required for PIP2 binding.

Highlights

vinculin tail domain (Vt)-(884 –1066) has previously been shown to bind to pure PS and PI vesicles at physiological ionic strength [27], we found that under physiological lipid and salt concentrations, no significant binding of Vt-(879 –1066) to PS or PI in mixed lipid vesicles was observed
The vinculin tail domain has been reported to interact with pure PI vesicles [27] and pure PS vesicles (using Vt-(879 –1066)) [26]
We found that changes in the 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of Vt⌬C are indicative of loss in structure relative to Vt, while Vt⌬C5, which removes only the hydrophobic hairpin at the C terminus of Vt, shows similar nuclear magnetic resonance spectroscopy (NMR) spectra to wild-type Vt, indicating a similar fold

Summary

The abbreviations used are

Vinculin tail domain; PIP2, phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; DPC, dodecylphosphocholine. Activation by acidic phospholipids would require a ligand lished studies have utilized constructs containing residues 811–. Given that multi- 1066, 858 –1066, 879 –1066, 881–1066, and 884 –1066 (26, 28, ple ligands have been proposed to play a role in vinculin activa- 40 – 42), with constructs containing residues 879 –1066 and tion, different combinations of ligands may allow vinculin acti- 884 –1066, extensively employed for characterizing Vt-ligand vation to be spatially and temporal regulated [30]. We will refer to Vt-(879 –1066), the their putative role in vinculin activation, acidic phospholipids construct used to determine the crystal structure of vinculin tail have been proposed to facilitate PKC-phosphorylation, domain as wild-type Vt, and the construct containing 884 –. (PI), and phosphatidylinositol 4,5-bisphosphate (PIP2), the relative affinity for various acidic phospholipids under conditions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION