Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.

Evangelos Papagrigoriou,Igor L Barsukov,Neil Bate,Alexandre R Gingras,Ian J Fillingham,David R Critchley,Jonas Emsley,Wolfgang H Ziegler,Ronald Frank,Gordon Ck Roberts,Bipin Patel

doi:10.1038/sj.emboj.7600285

Abstract

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482-789. Talin 482-655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656-789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482-655 fold. We demonstrate that the talin 482-655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

Highlights

Integrins, the principal family of adhesion molecules supporting cellular interactions with the extracellular matrix, are ab-heterodimers that exist in both high- and low-affinity states (Hynes, 2002)
The FERM domain binds to and activates the type 1 gamma isoform of PI4050-kinase, which is involved in focal adhesion assembly (Di Paolo et al, 2002; Ling et al, 2002), and the protein tyrosine kinase FAK (Chen et al, 1995), which is important in focal adhesion turnover and cell migration (Ilic et al, 1997)
We have identified the residues involved in vinculin binding, which align on the hydrophobic face of an amphipathic helix

Summary

Introduction

The principal family of adhesion molecules supporting cellular interactions with the extracellular matrix, are ab-heterodimers that exist in both high- and low-affinity states (Hynes, 2002). The short integrin ab cytodomains play a key role in affinity modulation, and more than 20 proteins that interact with these domains, largely the bsubunit, have been identified (Liu et al, 2000). These include talin, a-actinin, filamin, ILK and tensin, which are thought to Received: 20 April 2004; accepted: 3 June 2004; published online: 22 July 2004. The talin head contains an FERM domain (residues 86–400) with binding sites for several b-integrin cytodomains (Calderwood, 2004), as well as the cytodomain of the C-type lectin layilin, which colocalizes with talin in membrane ruffles (Borowsky and Hynes, 1998). The talin rod, which is dimeric, contains a second lower affinity integrin-binding site (residues 1984–2541) (Xing et al, 2001; Tremuth et al, 2004), a highly conserved C-terminal actin-binding site (residues 2345–2541) (Hemmings et al, 1996; McCann and Craig, 1997) and three binding sites for the cytoskeletal protein vinculin (Hemmings et al, 1996), which in turn has multiple binding partners including F-actin (Jockusch and Rudiger, 1996)

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Results

Conclusion