The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin ␤ tails contain one or two NPXY͞F motifs that can form ␤ turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-␣ bind to integrin ␤ tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin ␤ tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin ␣IIb␤3. Mutations of the integrin ␤ tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin ␤ tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain-and loss-of-function mutations in ␤7 and ␤3 tails. These data establish that short integrin ␤ tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism. I ntegrin adhesion receptors are heterodimers of ␣ and ␤ subunits, which combine to form a large extracellular domain, two transmembrane domains (one for each subunit), and a cytoplasmic domain typically composed of the short ␣ and ␤ C-terminal cytoplasmic tails (1). Bidirectional signal transduction through integrin adhesion receptors is essential for a wide variety of functions, including cell adhesion and migration, and assembly and remodeling of the extracellular matrix. Binding of intracellular proteins to integrin cytoplasmic tails is an important step in the transduction of signals to and from integrin-adhesion receptors (2). Integrin ␤ cytoplasmic tails, with the exception of those of ␤4 and ␤8, are short (Ͻ60 residues) and contain one or two NPXY or NPXY-like motifs (Fig. 1A), the first of which has the propensity to form a ␤ turn (3). Such ␤ turn-forming sequences frequently serve to bind to phosphotyrosine-binding (PTB) domains (4). NXXY motif-dependent binding of the Shc PTB domain to the large (Ͼ1,000 residues) ␤4 cytoplasmic tail has been observed (5) and molecular modeling studies suggested that the interaction of integrin cytoplasmic domain-associated protein (ICAP)1-␣ with ␤1A (6), and of talin with ␤3 (7), are mediated by PTB domain-like interactions. The solved crystal structure of a complex of a talin fragment with part of the ␤3 tail verified th...

Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.

Polo-like kinase (Plk1) is crucial for cell cycle progression through mitosis. Here we present the molecular and structural mechanisms that regulate the substrate recognition of Plk1 and influence its centrosomal localization and activity. Our work shows that Plk1 localization is controlled not only by the polo box domain (PBD); remarkably, the kinase domain is also involved in Plk1 targeting mechanism to the centrosome. The crystal structures of the PBD in complex with Cdc25C and Cdc25C-P target peptides reveal that Trp-414 is fundamental in their recognition regardless of its phosphorylation status. Binding measurements demonstrate that W414F mutation abolishes molecular recognition and diminishes centrosomal localization. Therefore, Plk1 centrosomal localization is not controlled by His-538 and Lys-540, the residues involved in phosphorylated target binding. The different conformations of the loop, which connects the polo boxes in the apo and the PBD-Cdc25C and PBD-Cdc25C-P complex structures, together with changes in the proline adjacent to the phosphothreonine in the target peptide, suggest a regulatory mechanism to detect binding of unphosphorylated or phosphorylated target substrates. Altogether, these data propose a model for the interaction between Plk1 and Cdc25C.cell cycle ͉ enzymes ͉ kinase ͉ protein structure ͉ polo box domain