Lucky kindlin: A cloverleaf at the integrin tail.

In a paper published in PNAS, Li et al. (1) solve the long-awaited crystal structure of kindlin-2, which plays a central role in integrin activation, clustering, and signaling (2, 3). Integrins are a large family of heterodimeric adhesion molecules composed of αand β-subunits and expressed on almost all cells. They mediate cell matrix adhesion by binding extracellular matrix proteins, such as collagens, fibronectin, and laminins, and cell–cell adhesion by interacting with counterreceptors such as VCAM, ICAM, and cadherins (Fig. 1A). Upon ligand binding, integrins cluster and assemble an enormous number of proteins at their short cytoplasmic domains, which are collectively called the adhesome (4). The adhesome transmits biochemical signals into the cell and connects integrins to the actin cytoskeleton, which, in turn, enables the transduction of myosin-II–generated traction forces to ligated integrins. The integrin α/β-subunits have large ectodomains that associate at their head domains to form the ligand-binding site, single-span transmembrane domains, and short cytoplasmic tails. A hallmark of integrins is that the affinity of their ligand-binding sites is tightly regulated by complex conformational changes affecting the entire integrin molecule: They reversibly shift from a low-affinity (or inactive) state, in which they adopt a bent-closed (BC) or extended-closed (EC) conformation, to the high-affinity (or active) state characterized by an extended-open (EO) conformation (2, 5) (Fig. 1A). The BC and EC conformations are characterized by a “closed” ligand-binding site, a loose connection between the transmembrane helices and cytoplasmic tails of αand β-subunits, and either an association of the ectodomain headpiece with the legs (BC) or an extension of the α/β-subunits (EC). The conversion of integrins into the EO conformation is characterized by the separation of the legs, transmembrane, and tail domains; the swing-away of the hybrid domain from the α-subunit; and the opening of the ligandbinding site, which results in a massive increase in affinity for ligand. A recent study measured the intrinsic affinity of α5β1 integrin on K562 cells for fibronectin and the free energy for each activation state of α5β1 (6). The measurements revealed that the affinity is 4,000-fold higher for the EO conformation compared with the EC or BC conformation. Furthermore, the free energy required to extend α5β1 is about 4 kcal/mol, indicating that extension is the major obstacle that needs to be overcome to activate α5β1 integrin (6). A vast body of evidence indicates that talin and kindlin induce the high-affinity state of integrins (7). Talins consist of two isoforms (talin-1 and talin-2) that are composed of an N-terminal FERM (protein 4.1, ezrin, radixin, moesin) and a C-terminal rod domain. The talin FERM domain is composed of classical F1, F2, and F3 modules and an extra, atypical F0 module. The F3 module contains a phosphotyrosine-binding motif that binds to the first (or membrane-proximal) of two conserved NxxYmotifs in β-integrin tails, as well as to two phenylalanine residues located in the juxtamembrane region (8, 9). The rod domain harbors multiple Vinculin and two F-actin–binding sites that link integrin to the actomyosin system. Interestingly, the crystal structure of the talin F0–F3 modules revealed an extended conformation rather than the compact cloverleaf-shaped structure that is typical for FERM domains (10, 11). The kindlin family consists of three isoforms (kindlin-1–kindlin-3), which are expressed in a tissue-specific manner (3, 7, 12). Kindlins consist of a FERM domain and a pleckstrin homology (PH) domain that is inserted into the F2 module. Biochemical interaction studies revealed that the F3 modules of kindlins bind to the membrane-distal NxxY motif; the adjacent threonine/serine residue; and, at least in integrin β1, the C-terminal carboxylate moiety (13). The prevailing view is that agonist-induced signaling (e.g., chemokine signaling) activates talin and kindlin, which then bind the integrin β-tails (also called inside-out signaling). Subsequently, talin activates integrins (EO state) by inducing the separation of the transmembrane domains of the αand β-subunits, whereas kindlin induces integrin clustering (3). However, integrins can also be activated upon binding to extracellular ligands, which cannot be explained with the current model. An alternative activation model for α5β1 binding to fibronectin, which would enable