The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations.

Integrins are heterodimeric (αß) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.

Conformational changes in talin on binding to anionic phospholipid membranes facilitate signaling by integrin transmembrane helices.

Integrins are heterodimeric (αß) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/ß integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/ß TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and ß transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.

Talins and kindlins: partners in integrin-mediated adhesion.

Integrin receptors provide a dynamic, tightly-regulated link between the extracellular matrix (or cellular counter-receptors) and intracellular cytoskeletal and signalling networks, enabling cells to sense and respond to their chemical and physical environment. Talins and kindlins, two families of FERM-domain proteins, bind the cytoplasmic tail of integrins, recruit cytoskeletal and signalling proteins involved in mechanotransduction and synergize to activate integrin binding to extracellular ligands. New data reveal the domain structure of full-length talin, provide insights into talin-mediated integrin activation and show that RIAM recruits talin to the plasma membrane, whereas vinculin stabilizes talin in cell-matrix junctions. How kindlins act is less well-defined, but disease-causing mutations show that kindlins are also essential for integrin activation, adhesion, cell spreading and signalling.

Characterization of 14-3-3-ζ Interactions with integrin tails.

Integrins are a family of heterodimeric (α+ß) adhesion receptors that play key roles in many cellular processes. Integrins are unusual in that their functions can be modulated from both outside and inside the cell. Inside-out signaling is mediated by binding adaptor proteins to the flexible cytoplasmic tails of the α- and ß-integrin subunits. Talin is one well-known intracellular activator, but various other adaptors bind to integrin tails, including 14-3-3-ζ, a member of the 14-3-3 family of dimeric proteins that have a preference for binding phosphorylated sequence motifs. Phosphorylation of a threonine in the ß2 integrin tail has been shown to modulate ß2/14-3-3-ζ interactions, and recently, the α4 integrin tail was reported to bind to 14-3-3-ζ and associate with paxillin in a ternary complex that is regulated by serine phosphorylation. Here, we use a range of biophysical techniques to characterize interactions between 14-3-3-ζ and the cytoplasmic tails of α4, ß1, ß2 and ß3 integrins. The X-ray structure of the 14-3-3-ζ/α4 complex indicates a canonical binding mode for the α4 phospho-peptide, but unexpected features are also observed: residues outside the consensus 14-3-3-ζ binding motif are shown to be essential for an efficient interaction; in contrast, a short ß2 phospho-peptide is sufficient for high-affinity binding to 14-3-3-ζ. In addition, we report novel 14-3-3-ζ/integrin tail interactions that are independent of phosphorylation. Of the integrin tails studied, the strongest interaction with 14-3-3-ζ is observed for the ß1A variant. In summary, new insights about 14-3-3-ζ/integrin tail interactions that have implications for the role of these molecular associations in cells are described.

Structural analysis of collagen type I interactions with human fibronectin reveals a cooperative binding mode.

Despite its biological importance, the interaction between fibronectin (FN) and collagen, two abundant and crucial tissue components, has not been well characterized on a structural level. Here, we analyzed the four interactions formed between epitopes of collagen type I and the collagen-binding fragment (gelatin-binding domain (GBD)) of human FN using solution NMR, fluorescence, and small angle x-ray scattering methods. Collagen association with FN modules (8-9)FnI occurs through a conserved structural mechanism but exhibits a 400-fold disparity in affinity between collagen sites. This disparity is reduced in the full-length GBD, as (6)FnI(1-2)FnII(7)FnI binds a specific collagen epitope next to the weakest (8-9)FnI-binding site. The cooperative engagement of all GBD modules with collagen results in four broadly equipotent FN-collagen interaction sites. Collagen association stabilizes a distinct monomeric GBD conformation in solution, giving further evidence to the view that FN fragments form well defined functional and structural units.

Biophysical analysis of Kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal ß-subunit NPXY motif.

Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and ß-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the ß(1A) and ß(1D) isoforms, although the interaction is stronger with ß(1A). An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/ß-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended.

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16-21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form more intricate superstructures. The crystal structure of filamin A domains 19-21 (FLNa19-21) revealed that domains 20 and 21 fold together and that the domain interaction can be autoregulatory. The solution structure of domains 18-19 showed a similar domain interaction, whereas domain pair 16-17 has a completely different domain packing mode. In this study, we characterize the domain organization of the FLNa domain sextet 16-21 using NMR spectroscopy. A structure model of this 60-kDa protein has been built using residual dipolar coupling restraints. RDCs and (15)N relaxation data have been used to characterize interdomain motions.

A helix heterodimer in a lipid bilayer: prediction of the structure of an integrin transmembrane domain via multiscale simulations.

Dimerization of transmembrane (TM) α helices of membrane receptors plays a key role in signaling. We show that molecular dynamics simulations yield models of integrin TM helix heterodimers, which agree well with available NMR structures. We use a multiscale simulation approach, combining coarse-grained and subsequent atomistic simulation, to model the dimerization of wild-type (WT) and mutated sequences of the αIIb and ß3 integrin TM helices. The WT helices formed a stable, right-handed dimer with the same helix-helix interface as in the published NMR structure (PDB: 2K9J). In contrast, the presence of disruptive mutations perturbed the interface between the helices, altering the conformational stability of the dimer. The αIIb/ß3 interface was more flexible than that of, e.g., glycophorin A. This is suggestive of a role for alternative packing modes of the TM helices in transbilayer signaling.

Multiscale simulations suggest a mechanism for integrin inside-out activation.

Integrins are large cell-surface adhesion receptors that can be activated to a high affinity state by the formation of an intracellular complex between the integrin ß-subunit tail, the membrane, and talin. The F2 and F3 subdomains of the talin head play a key role in formation of this complex. Here, activation of the integrin αIIb/ß3 dimer by the talin head domain was probed using multiscale molecular dynamics simulations. A number of novel insights emerge from these studies, including (i) the importance of the integrin αIIb subunit F992 and F993 residues in stabilizing the "off" state of the αIIb/ß3 dimer, (ii) a crucial role for negatively charged groups in the F2-F3/membrane interaction, (iii) binding of the talin F2-F3 domain to negatively charged lipid headgroups in the membrane induces a reorientation of the ß transmembrane (TM) domain, (iv) an increase in the tilt angle of the ß TM domain relative to the bilayer normal helps to destabilize the α/ß TM interaction and promote a scissor-like movement of the integrin TM helices. These results, combined with various published experimental observations, suggest a model for the mechanism of inside-out activation of integrins by talin.

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last ß-strand of domain 19, FLNa(19), and the first ß-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking ß-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Integrin structure, activation, and interactions.

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.

The tail of integrin activation.

Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation.

Structural diversity in integrin/talin interactions.

The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the ß-integrin subunit. Structural studies of this interaction have hitherto largely focused on the ß3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between ß1A, ß1D, and ß3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/ß tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/ß1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the ß tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.

The talin FERM domain is not so FERM.

The structure of the head domain of talin, an intracellular activator of integrin membrane adhesion receptors, has been solved by Elliott et al. (2010). A FERM domain can be identified in the head from sequence comparisons but, rather than having a compact structure of three subdomains, it has linear arrangement of four subdomains.

The structure of the talin/integrin complex at a lipid bilayer: an NMR and MD simulation study.

Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin ß cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the α/ß transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions.

The streptococcal binding site in the gelatin-binding domain of fibronectin is consistent with a non-linear arrangement of modules.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the (8)F1(9)F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to (8)F1(9)F1 and that UR binding to (8)F1 is likely to occur through anti-parallel ß-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem ß-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.

Implications for collagen binding from the crystallographic structure of fibronectin 6FnI1-2FnII7FnI.

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, (6)FnI(1-2)FnII(7-9)FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains (8-9)FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains (6)FnI(1-2)FnII(7)FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains (2)FnII and (7)FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the (8-9)FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

The structure of an integrin/talin complex reveals the basis of inside-out signal transduction.

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.

Beta integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation.

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the beta integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated (15)N-labeled beta3, beta1A, and beta7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of beta3, beta1A, and beta7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of beta integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.