Two Distinct Head-Tail Interfaces Cooperate to Suppress Activation of Vinculin by Talin

Daniel M Cohen,Hui Chen,Robert P Johnson,Begum Choudhury,Susan W Craig

doi:10.1074/jbc.m414704200

Daniel M Cohen, Hui Chen + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m414704200

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Abstract

Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (Vt) and residues 1-258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (Vh residues 710-836) and Vt. Charge-to-alanine mutations in Vt revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their Kd values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact Vt yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in Kd values between Vt binding to V-(1-258), as opposed to full-length Vh-(1-851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length Vh, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-Vt interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-Vt interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-Vt and D4-Vt interfaces provide the high affinity (approximately 10(-9)) autoinhibition observed in full-length vinculin.

Highlights

Quantitative Dissection of the Head-Tail Interaction Surface—The clustering of strong head-tail interaction mutations at the base of Vt is unexpected given the recently proposed structural model for the autoinhibitory head-tail interaction, in which the top of the Vt bundle exclusively contacts the D1 domain in vinculin head, and this interaction is proposed to account for the high affinity, autoinhibited state of vinculin
Solution phase assay to measure Kd values for head-tail interaction, using a fluorescent supernatant depletion of YFP-tagged Vh by His-tagged Vt
It has been reported that D1 binds Vt with nanomolar affinity in a solid-phase binding assay [33], such assays introduce systemic error in Kd measurement by failing to account for the high local concentration effect of ligand immobilization

Summary

The abbreviations used are

V, vinculin; Vt, vinculin tail domain residues 884 –1066; Vh, vinculin head domain residues 1– 851; D1, vinculin residues 1–258; D4, vinculin residues 710 – 836; F-actin, filamentous actin; VBS3, talin residues 1945–1970; YFP, yellow fluorescent protein; GFP; green fluorescent protein; PIP2, phosphatidylinositol 4,5bisphosphate; NTA, nitrilotriacetic acid; WT, wild type; Bicine, N,Nbis(2-hydroxyethyl)glycine; DMEM, Dulbecco’s modified Eagle’s medium; HEK, human embryonic kidney cells. Whereas mechanistic insights into the molecular basis of these phenotypes are limited with a few notable exceptions [7, 8], the aforementioned effects are generally consistent with the idea that vinculin functions as a mechanical linker between the plasma membrane and actin cytoskeleton through cross-linking several adhesion proteins This model derives from in vitro studies that identified interactions between vinculin and numerous focal adhesions proteins including talin [9], ␣-actinin [10, 11], paxillin [12], VASP [13], vinexin/ponsin family members [14, 15], and F-actin [16, 17]. Recent crystallographic studies on the autoinhibited conformation of vinculin have revealed that the principal binding site for head-tail interaction is comprised of a large hydrophobic interface between the D1 domain of vinculin (residues 1–258) and the top of the five helix bundle structure comprising the vinculin tail (20 –22) This interface is disrupted upon binding of a talin-derived peptide (VBS3) to the D1 domain. This analysis redefines the autoinhibitory Vh domain as D1-D4, rather than D1 [20] or D1-D3 [22], and enables us to provide a detailed model for autoinhibition that accounts for the functional insufficiency of talin for activating native vinculin [22]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION