C OMMUNICATION Crystal Structure of the Talin Integrin Binding Domain 2 Tsz Ying Sylvia Cheung 1,2 , Michael J. Fairchild 1 , Raz Zarivach 3 , Guy Tanentzapf 1 ⁎ and Filip Van Petegem 2 ⁎ 1 Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada V6T 1Z3 2 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada V6T 1Z3 3 Department of Life Sciences, Ben-Gurion University, 84120 Beer-Sheva, Israel Received 24 December 2008; received in revised form 23 January 2009; accepted 23 January 2009 Available online 31 January 2009 Integrins are transmembrane receptors that mediate cell adhesion to the extracellular matrix and play essential roles in tissue development and maintenance. The cytoplasmic segment of integrin associates with talin, a large intracellularprotein thatlinks integrin to the actin cytoskeleton. Binding of talin via an integrin binding segment (IBS1)results in large conformational changesin the extracellularportion of integrin,which modulates the affinity of integrins for their extracellular matrix ligands. However,integrin binding also requires a second segment of talin (IBS2). Despite detailed descriptions of the integrin–IBS1 binding, the molecular determinants that drive the integrin–IBS2 association are poorly understood. Here, we describe the crystal structure of the talin IBS2 domain, which forms a five-helix bundle. The large structural homology with a vinculin binding domain hints at an ancient gene duplication and suggests that helix 4 may bind to vinculin if the bundle is unfolded. Mapping previous mutations on the surface highlights a likely binding interface for integrin. © 2009 Published by Elsevier Ltd. Edited by I.Wilson Keywords: talin;integrin;X-ray crystallography; structure; cell adhesion Cell adhesion to the extracellular matrix (ECM) is dependenton the integrin family of adhesion receptors.Integrinsare heterodimericmembrane proteins consisting of α and β subunits. They bind to ECM ligands on the outside of the cell and on the inside bind to cytoplasmic linkers that connect them to the actin cytoskeleton. 1 Integrin-mediated attach- ment to the ECM is essential for a wide variety of biologically important events and is implicated in pathological disorders such as skin blistering dis- eases, muscle degeneration, thrombosis, blood clot- ting disorders, and cancer. 2–5 The large cytoplasmic protein talin is a key linker between integrins and the cytoskeleton. 6,7 Talin binds to the intracellular tail of β integrins and connects it to actin either directly through its actin binding site or indirectly through other actin binding proteins such as vinculin. 6,7 Structural studies have described thelinkage between integrin and the ECM in detail, 8–10 and recent progress has been made in investigating the talin–integrin association. 11 Talin contains two con- served integrin binding sites (IBSs), an N-terminal IBS1 and a C-terminal IBS2. Full-length talin (∼ 2500 aa) is composed ofa 50-kDa head and a 200-kDa rod. 7 The talin head is formed by a FERM (band 4.1, ezrin, radixin, moesin) domain, consisting of three subdomains (F1, F2, and F3).The IBS1 is found in the F3 subdomain and has structural homology to phosphotyrosinebinding domains, well-characterized domains that are known to bind NPxY motifs. 12,13 Most integrin β subunits contain two well-conserved NPxY motifs within their ∼ 47- residue cytoplasmic tail, and it is the β subunit within the integrin αβ heterodimer that forms the primary link to the cytoskeleton.Both crystallo- graphic and NMR studies confirmed the direct *Corresponding authors. G. Tanentzapf is to be contacted at Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada V6T 1Z3. F. V. Petegem, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada V6T 1Z3. E-mail addresses: tanentz@interchange.ubc.ca; Petegem@interchange.ubc.ca. Abbreviations used: ECM, extracellular matrix; FERM domain, band 4.1, ezrin, radixin, moesin domain; IBS, integrin binding site; VBS, vinculin binding site; PDB, Protein Data Bank. doi:10.1016/j.jmb.2009.01.053 J. Mol. Biol. (2009) 387, 787–793 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Published by Elsevier Ltd.