The Structure of the N-Terminus of Kindlin-1: A Domain Important for αIIbβ3 Integrin Activation

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced αIIbβ3 integrin activation and for the localization of kindlin-1 to focal adhesions.     

Crystal structure of kindlin-2 PH domain reveals a conformational transition for its membrane anchoring and regulation of integrin activation

Kindlin-2 belongs to a subfamily of FERM domain containing proteins, which plays key roles in activating integrin transmembrane receptors and mediating cell adhesion. Compared to conventional FERM domains, kindlin-2 FERM contains an inserted pleckstrin homology (PH) domain that specifically binds to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and regulates the kindlin-2 function. We have determined the crystal structure of kindlin-2 PH domain at 1.9 ? resolution, which reveals a conserved PH domain fold with a highly charged and open binding pocket for PIP3 head group. Structural comparison with a previously reported solution structure of kindlin-2 PH domain bound to PIP3 head group reveals that upon PIP3 insertion, there is a significant conformational change of both the highly positively charged loop at the entry of the PIP3 binding pocket and the entire β barrel of the PH domain. We propose that such “induced-fit” type change is crucial for the tight binding of PIP3 to anchor kindlin-2 onto the membrane surface, thereby promoting its binding to integrins. Our results provide important structural insight into kindlin-2-mediated membrane anchoring and integrin activation.     

A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin β tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin β subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in αIIbβ3 integrin activation, but not for kindlin binding to integrin β tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well β-integrin tails contribute to the ability of kindlins to activate integrins.     

NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain

Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using ¹⁵N-¹H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property.     

Kindlins, integrin activation and the regulation of talin recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.     

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P₃; K_D ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P₃ as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P₂ on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P₃ is affected by the presence of PO₄ ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.     

Kindlin-1 and -2 Directly Bind the C-terminal Region of �� Integrin Cytoplasmic Tails and Exert Integrin-specific Activation Effects

Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Talin binding to integrin β tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin β tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin β tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr⁷⁹⁵), a preceding threonine-containing region (Thr⁷⁸⁸ and Thr⁷⁸⁹) of the integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp⁶¹² and kindlin-2 Trp⁶¹⁵) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin β3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous α5β1 or stably expressed αIIbβ3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate αIIbβ3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate β1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.Integrins are a family of αβ heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. A key feature of integrins is their ability to dynamically regulate their affinity for extracellular ligands. In a tightly regulated process generally termed integrin activation, intracellular signals that impinge upon the β subunit cytoplasmic tail induce conformational rearrangements in the integrin extracellular domains, increasing the binding affinity for extracellular ligands (1-3). Ligand-bound integrins then recruit additional signaling, adaptor, and cytoskeletal proteins to the integrin cytoplasmic domains, providing mechanical connections to the actin cytoskeleton and a link to a variety of signal transduction pathways (2-8).Recent years have seen significant advances in our understanding of integrin activation. Notable among these is the identification of the actin- and integrin-binding protein talin as a key integrin activator (1, 9). The 50-kDa talin head contains the principal integrin-binding site, and expression of the talin head is sufficient to activate β1 and β3 integrins (10, 11). The talin head contains a FERM (four point one ezrin radixin moesin) domain. FERM domains consist of trefoil arrangement of three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3 subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in integrin β tails, and this interaction is necessary for integrin activation in vitro and in vivo (10, 12-19). However, although abundant evidence supports the importance of talin binding to integrin β tails during integrin activation, differences in sensitivity of integrins to talin activation and submaximal activation by overexpressed talin suggested that other activating factors may cooperate with talin (10, 20). In an attempt to identify and characterize potential co-activators, we investigated the kindlin family of FERM domain-containing proteins.Kindlin family proteins (21) were first characterized in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112, was identified in an embryonic screen for defective motility and shown to be essential for the assembly of proper cell-matrix adhesion structures, where it normally co-localized with β integrin (22-24). UNC-112 is conserved across many species, because the nematode, fly, and human homologs are ∼60% similar (∼41% identical) over their entire length (24). Humans express three known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2 (Mig2 and mig-2), and kindlin-3 (Mig2B and URP2) (25-27). Kindlin-1 and -2 are most closely related, sharing 60% identity and 74% similarity, whereas kindlin-3 shares 53% identity and 69% similarity to kindlin-1 and 49% identity and 67% similarity to kindlin-2 (28). The kindlin proteins all contain a predicted Pleckstrin homology domain and a FERM domain that is most closely related to the talin FERM domain, particularly within the integrin-binding F3 subdomain (29). Based on this sequence similarity we proposed that kindlin FERM domains may directly bind integrin β tails, and we previously showed that kindlin-1 could be pulled down from cell lysates using recombinant integrin β1 and β3 tails and that kindlin-1 co-localized with integrins in focal adhesions (29). A similar localization was reported for kindlin-2 (26, 30), and recent reports provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of integrin activation (31-33). Here, we have used integrin pulldown assays to demonstrate direct binding of full-length kindlin-1 to the cytoplasmic tails of β1A and β3 integrins and to identify key binding residues within the integrin tails and the kindlin F3 subdomain. We confirm that these interactions are important for recruiting kindlin-1 to focal adhesions and show that, contrary to expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3 integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate with expressed talin head to activate β3 but not β1 integrins. We therefore provide the first data suggesting that kindlin-1 and -2 effects on integrin activation may show β subunit specificity.     

Phosphatidylinositol phosphate kinase type 1gamma and beta1-integrin cytoplasmic domain bind to the same region in the talin FERM domain

Talin is an essential component of focal adhesions that couples beta-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin beta3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1gamma (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The Kd for the interaction determined by Trp fluorescence was 6 microm, and the R358A mutation increased the Kd to 35 microm. Comparison of these results with those of the crystal structure of a beta3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin beta1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer.     

Kindlin Assists Talin to Promote Integrin Activation

Integrin αIIbβ3 is a predominant type of integrin abundantly expressed on the surface of platelets and its activation regulates the process of thrombosis. Talin and kindlin are cytoplasmic proteins that bind to integrin and modulate its affinity for extracellular ligands. Although the molecular details of talin-mediated integrin activation are known, the mechanism of kindlin involvement in this process remains elusive. Here, we demonstrate that the interplay between talin and kindlin promotes integrin activation. Our all-atomic molecular dynamics simulations on complete transmembrane and cytoplasmic domains of integrin αIIbβ3, talin1 F2/F3 subdomains, and the kindlin2 FERM domain in an explicit lipid-water environment over a microsecond timescale unraveled the role of kindlin as an enhancer of the talin interaction with the membrane proximal region of β−integrin. The cooperation of kindlin with talin results in a complete disruption of salt bridges between R995 on αIIb and D723/E726 on β3. Furthermore, kindlin modifies the molecular mechanisms of inside-out activation by decreasing the crossing angle between transmembrane helices of integrin αIIbβ3, which eventually results in parallelization of integrin dimer. In addition, our control simulation featuring integrin in complex with kindlin reveals that kindlin binding is not sufficient for unclasping the inner-membrane and outer-membrane interactions of integrin dimer, thus ruling out the possibility of solitary action of kindlin in integrin activation.     

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

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) – a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic “pull-push” process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic “steric clash” model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.     

Kindlin-3 mediates integrin αLβ2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1)

Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLβ2 outside-in signaling. It also promotes micro-clustering of integrin αLβ2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLβ2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLβ2 outside-in signaling.     

Spatial coordination of kindlin-2 with talin head domain in interaction with integrin β cytoplasmic tails

Both talin head domain and kindlin-2 interact with integrin β cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to β cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin β tails. Kindlin-2 interacted well with β(1) and β(3) tails but poorly with the β(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the β(3) tail, and the sequence in β(2) was non-permissive. The region at the C termini of integrin β(1) and β(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin β(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/β tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.     

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

Membrane binding of the N-terminal ubiquitin-like domain of kindlin-2 is crucial for its regulation of integrin activation

Kindlin-2 belongs to an emerging class of regulators for heterodimeric (α/β) integrin adhesion receptors. By binding to integrin β cytoplasmic tail via its C-terminal FERM-like domain, kindlin-2 promotes integrin activation. Intriguingly, this activation process depends on the N terminus of kindlin-2 (K2-N) that precedes the FERM domain. The molecular function of K2-N is unclear. We present the solution structure of K2-N, which displays a ubiquitin fold similar to that observed in kindlin-1. Using chemical shift mapping and mutagenesis, we found that K2-N contains a conserved positively charged surface that binds to membrane enriched with negatively charged phosphatidylinositol-(4,5)-bisphosphate. We show that while wild-type kindlin-2 is capable of promoting integrin activation, such ability is significantly reduced for its membrane-binding defective mutant. These data suggest a membrane-binding function of the ubiquitin-like domain of kindlin-2, which is likely common for all kindlins to promote their localization to the plasma membrane and control integrin activation.     

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages—but none of the premetazoans—had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament–associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness.     

Kindlin-2 (Mig-2): a co-activator of beta3 integrins

Integrin activation is essential for dynamically linking the extracellular environment and cytoskeletal/signaling networks. Activation is controlled by integrins' short cytoplasmic tails (CTs). It is widely accepted that the head domain of talin (talin-H) can mediate integrin activation by binding to two sites in integrin beta's CT; in integrin beta(3) this is an NPLY(747) motif and the membrane-proximal region. Here, we show that the C-terminal region of integrin beta(3) CT, composed of a conserved TS(752)T region and NITY(759) motif, supports integrin activation by binding to a cytosolic binding partner, kindlin-2, a widely distributed PTB domain protein. Co-transfection of kindlin-2 with talin-H results in a synergistic enhancement of integrin alpha(IIb)beta(3) activation. Furthermore, siRNA knockdown of endogenous kindlin-2 impairs talin-induced alpha(IIb)beta(3) activation in transfected CHO cells and blunts alpha(v)beta(3)-mediated adhesion and migration of endothelial cells. Our results thus identify kindlin-2 as a novel regulator of integrin activation; it functions as a coactivator.     

Structural determinants of integrin recognition by talin

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.     

Interactions of the pleckstrin homology domain with phosphatidylinositol phosphate and membranes: characterization via molecular dynamics simulations

The mechanism of interaction of pleckstrin homology (PH) domains with phosphatidylinositol 4,5-bisphosphate (PIP 2)-containing lipid bilayers remains uncertain. While crystallographic studies have emphasized PH-inositol 1,4,5-trisphosphate (IP 3) interactions, biophysical studies indicate a degree of less specific protein-bilayer interactions. We have used molecular dynamics simulations to characterize the interactions of the PH domain from phospholipase C-delta1 with IP 3 and with PIP 2, the latter in lipid bilayers and in detergent micelles. Simulations of the PH domain in water reveal a reduction in protein flexibility when IP 3 is bound. Simulations of the PH domain bound to PIP 2 in lipid bilayers indicate a tightening of ligand-protein interactions relative to the PH-IP 3 complex, alongside formation of H-bonds between PH side chains and lipid (PC) headgroups, and a degree of penetration of hydrophobic side chains into the core of the bilayer. Comparison with simulations of the PH-bound domain to a PC bilayer in the absence of PIP 2 suggests that the presence of PIP 2 increases the extent of PH-membrane interactions. Thus, comparative molecular dynamics simulations reveal how a PI-binding domain undergoes changes in conformational dynamics on binding to a PIP 2-containing membrane and how interactions additional to those with the PI headgroup are formed.     

The Structure of the talin head reveals a novel extended conformation of the FERM domain

FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.     

The phosphotyrosine binding-like domain of talin activates integrins

Cellular regulation of the ligand binding affinity of integrin adhesion receptors (integrin activation) depends on the integrin beta cytoplasmic domains (tails). The head domain of talin binds to several integrin beta tails and activates integrins. This head domain contains a predicted FERM domain composed of three subdomains (F1, F2, and F3). An integrin-activating talin fragment was predicted to contain the F2 and F3 subdomains. Both isolated subdomains bound specifically to the integrin beta3 tail. However, talin F3 bound the beta3 tail with a 4-fold higher affinity than talin F2. Furthermore, expression of talin F3 (but not F2) in cells led to activation of integrin alpha(IIb)beta3. A molecular model of talin F3 indicated that it resembles a phosphotyrosine-binding (PTB) domain. PTB domains recognize peptide ligands containing beta turns, often formed by NPXY motifs. NPX(Y/F) motifs are highly conserved in integrin beta tails, and mutations that disrupt this motif interfere with both integrin activation and talin binding. Thus, integrin binding to talin resembles the interactions of PTB domains with peptide ligands. These resemblances suggest that the activation of integrins requires the presence of a beta turn at NPX(Y/F) motifs conserved in integrin beta cytoplasmic domains.