The Structure of an Interdomain Complex That Regulates Talin Activity

Abstract

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316 -326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with ␤3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1) and in coupling integrins to the actin cytoskeleton (2). Thus, depletion of talin results in defects in integrin activation (3), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4). In the whole organism, studies on the single talin gene in worms (5) and flies (6) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8, 9), defects in the membrane-cytoskeletal interface in megakaryocytes (10), and disruption of the myotendinous junction in skeletal muscle (11). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12).Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (ϳ50 kDa) linked to an extended flexible rod (ϳ220 kDa). The talin head contains a FERM 2 domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2). Studies by Wegener et al. (30) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin ␤-subunits (13). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin ␣-and ␤-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activati...

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

“…Notably, (i) the carboxyl group of E1770 of talin-RS forms a buried salt-bridge with K318-NH 3 + and a strong hydrogen bond with the backbone of G321, and van der Waals contact with the backbone of M319 of talin-F3; (ii) the hydroxyl group of T1767 hydrogen bonds with K318 side chain -NH 3 + , N323 side chain -NH 2 , and backbone carbonyl of K322, respectively, and the methyl group of T1767 also makes van der Waals contacts with G321 and N323, respectively; (iii) the side chain of M1802 exhibits hydrophobic interac- The autoinhibitory interface of the crystal structure is consistent with the previously reported result that the L325R mutation had no effect on the complex formation [19], since L325 is not in the complex interface ( Figure 1C). It is also consistent with extensive loss-offunction mutagenesis data [19,20], including mutations of M319A, S365D, S379R, Q381V, T1767A/D/E, and E1770A, which all perturb the interface environment. Specifically, M319A, T1767A/D/E, and E1770A directly disrupt the interface based on the above described interactions.…”

“…The structure reveals an unexpected dual inhibitory topology in which talin-RS not only sterically masks the integrinbinding site on talin-FERM via one large interface, but also electrostatically hinders the membrane-targeting of talin-FERM via another extensively negatively charged surface. Such a topology is consistent with the biochemical and functional data, but strikingly different from a previous model [20]. The topology further suggests a "pull-push" mechanism for talin activation by membrane enriched with PIP2, which differs distinctly from the classic steric-clash mechanism for the conventional PIP2-mediated activation of FERM proteins activation.…”

“…The crystal structure is drastically different from the previously reported NMR-based model that was primarily built by the HADDOCK program using ambiguous chemical shift constraints [20]. Overlay of talin-F3 between the crystal structure and the HADDOCK model reveals that the bound talin-RS in the crystal structure is rotated nearly 90° versus the model (Supplementary information, Figure S1D).…”

“…Talin-F2F3 and talin-F3 bind to talin-RS identically as judged by NMR, indicating that F2 does not affect the talin-F3/talin-RS interface (Supplementary information, Figure S1A). In the complex, talin-F2F3 and talin-RS exhibit the same fold as their free forms [20,31], but pack extensively with a buried interface of 1750 Å 2 where the five-helix bundle of talin-RS is held in place by two protruding fingers of talin-F3 ( Figure 1A). Superposition of talin-F2F3 bound to talin-RS with that bound to the integrin β1 CT [13] (PDB code 3G9W) shows that a major portion of membrane-proximal region but not the membrane-distal region of integrin β1 CT is sterically blocked by the talin-F2F3/talin-RS interface ( Figure 1B).…”

“…Talin can adopt inactive and active states during dynamic cell adhesion events, but the underlying molecular basis for this transition is elusive [2]. Previous studies suggested that the inactive talin has an autoinhibited conformation, where a key integrin-binding site on talin-F3 is self-masked by a talin-R segment (talin-RS) [19,20]. Membrane lipid phosphotidylinositol-4,5-bisphosphate (PIP2) [19,21], which is locally enriched by talin-recruited PIPKIγ kinase [22,23], was shown to activate talin [19,21] and promote integrin-mediated cell adhesion [15,[24][25][26][27][28], but the detailed structural basis of this is not clear.…”