title: Structural determinants of integrin binding to the talin rod.
abstract: The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.
authors.affiliation: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Gingras
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Ziegler
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Bobkov
authors.forename: M Gordon
authors.initials: MG
authors.lastname: Joyce
authors.forename: Domenico
authors.initials: D
authors.lastname: Fasci
authors.forename: Mirko
authors.initials: M
authors.lastname: Himmel
authors.forename: Sven
authors.initials: S
authors.lastname: Rothemund
authors.forename: Anett
authors.initials: A
authors.lastname: Ritter
authors.forename: J Günter
authors.initials: JG
authors.lastname: Grossmann
authors.forename: Bipin
authors.initials: B
authors.lastname: Patel
authors.forename: Neil
authors.initials: N
authors.lastname: Bate
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Goult
authors.forename: Jonas
authors.initials: J
authors.lastname: Emsley
authors.forename: Igor L
authors.initials: IL
authors.lastname: Barsukov
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
authors.forename: Robert C
authors.initials: RC
authors.lastname: Liddington
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
authors.forename: David R
authors.initials: DR
authors.lastname: Critchley
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016608:Talin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016023:Integrins; D016608:Talin; C496077:Tln1 protein, mouse; D016596:Vinculin