Three-Dimensional Structure of Vinculin Bound to Actin Filaments

Abstract

Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. We used a combination of electron microscopy, computational docking, and biochemistry to provide an atomic model of how the vinculin tail binds actin filaments. The vinculin tail actin binding site comprises two distinct regions. One of these regions is exposed in the full-length autoinhibited conformation of vinculin, whereas the second site is sterically occluded by vinculin's N-terminal domain. The partial accessibility of the F-actin binding site in the autoinhibited full-length vinculin structure suggests that F-actin can act as part of a combinatorial input framework with other binding partners such as alpha-catenin or talin to induce vinculin head-tail dissociation, thus promoting vinculin activation. Furthermore, binding to F-actin potentiates a local rearrangement in the vinculin tail that in turn promotes vinculin dimerization and, hence, formation of actin bundles.

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

“…Bundling and Barbed End Capping-Vt is known to crosslink actin filaments into bundles (11)(12)(13). We confirmed the bundling activity of Vt by using both a low speed sedimentation assay and a direct observation in TIRF microscopy (supplemental Fig.…”

“…4A). Vt binds F-actin and crosslinks actin filaments into bundles (11)(12)(13). The protein is regulated by an intramolecular interaction in which Vh interacts with Vt (14).…”

“…The C-terminal Arm of Vinculin Tail Is Required for Actin Filament Barbed End Capping but Not for Side Binding-Interestingly, the combination of cryoelectron microscopy reconstitution, probability of binding, and mutagenesis showed that the F-actin-binding domain located in Vt is divided into two spatially distinct clusters that contact two consecutive actin subunits (11,13). The lower patch, proximal to the barbed end, includes residues of helices 3 and 4 and the C-terminal arm following helix 5 of the tail domain.…”

“…The lower patch, proximal to the barbed end, includes residues of helices 3 and 4 and the C-terminal arm following helix 5 of the tail domain. The upper patch contains residues of helices 2 and 3 (13). We designed C-terminal deletions of the tail domain to remove the residues of the lower patch.…”

Single-particle reconstruction from EM images of helical filaments

“…A specific algorithm for single-particle type reconstruction of helical filaments was published in 2000 [12], and applications of this method have now been published in more than 35 papers. While many of these papers come from my own laboratory, an increasing number of applications are coming from other groups [13][14][15][16][17][18][19][20][21]. The algorithm, called the Iterative Helical Real Space Reconstruction (IHRSR) method, takes advantage of the helical symmetry present in images of helical filaments to impose helical symmetry on asymmetric reconstructions.…”