Structural basis of the migfilin-filamin interaction and competition with integrin beta tails

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro(5) and Pro(19) and binds to the CD face of the IgFLNa21 beta-sandwich. This interaction is similar to the previously characterized beta 7 integrin-IgFLNa21 interaction and migfilin and integrin beta tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.Actin cytoskeleton is a dynamic network that is involved in many fundamental cellular processes such as cell differentiation, morphology, endocytosis, exocytosis, cytokinesis, and cell movement. These events are regulated by proteins that interact with monomeric and filamentous actin. Filamins are actin filament-binding and cross-linking proteins. Filamin A and filamin B are both ubiquitously expressed, and their mutations in human patients cause developmental abnormalities in brain, cartilage, bones, and epithelial tissues (1). Filamin C is muscle-specific, and mutations thereof cause myofibrillar myopathy (2). Mice with targeted deletion of any of the filamin genes die either during development or soon after birth (3–6). These phenotypes are thought to reflect the roles of filamins as scaffolds of signaling pathways required for cell differentiation, regulators of cell migration, and stabilizers of cytoskeleton and cell membranes (1, 7).Filamins bind to actin filaments mainly via their N-terminal actin-binding domains and interact with other proteins via the 24 filamin type immunoglobulin-like domains (IgFLN),³ also called filamin repeats (8). Especially the C-terminal IgFLNs 16–24 contain several protein-protein interaction sites (1). Our previous structural studies have revealed that many proteins interact with filamins by forming an additional β-strand next to strand C of an individual IgFLN. The platelet von Willebrand factor receptor, glycoprotein (GP) Ibα, interacts in this way with IgFLNa17 (9). The integrin family adhesion receptor β subunits interact with IgFLNa21 and to a lesser extent with IgFLNa19 (10, 11). Furthermore, some signaling proteins use a similar interaction mode: the adaptor protein migfilin interacts with IgFLNa21 (12), and the Rho family GTPase-activating protein FilGAP interacts with IgFLNa23 (13, 14).Although structural details are known from many filamin interactions, it is not completely clear how these interactions are regulated. In some cases the regulation involves competition between multiple binding partners (10, 11). Alternative splicing (15), proteolysis of filamin (16–18), and ligand phosphorylation (11) also contribute to the regulation. Recently, it has become apparent that conformational changes in filamins may also be involved. For instance, actomyosin contraction exposes hidden cysteine residues in filamins (19). This opens the possibility that forces transmitted through actin filament may open up binding sites, and filamin may thus be involved in mechanosensor signaling.We have recently found a structural mechanism by which mechanical forces could regulate interactions at the C-terminal part of filamin. Our recent crystal structure revealed that IgFLNa20 forms a compact pair with IgFLNa21, and in this pair the N-terminal part of IgFLNa20 masks the integrin-binding site on IgFLNa21 (15). It is possible that this masking could be released by mechanical forces. Four lines of evidence led us to hypothesize that in addition to the IgFLNa20–21 pair, other similar domain pairs could exist at the C terminus of filamin: (i) the overall structure of the C-terminal part (IgFLNs 16–24) of filamin is relatively more compact than the N-terminal part of the molecule (IgFLNs 1–15) (8); (ii) the N-terminal sequences of even-numbered domains 16, 18, and 20 differ from other IgFLNs (20) (sequence alignment is shown in supplemental Fig. S1); (iii) in single-domain solution NMR structures of IgFLNc16, IgFLNb16, 18, and 20, the N-terminal part is not folded with the rest of the domain; and (iv) according to biochemical experiments, IgFLNa18 masks integrin binding to IgFLNa19 (15). We report here small angle x-ray scattering (SAXS) analysis showing that IgFLNa16–17 and 18–19 have overall dimensions very similar to those of the previously known domain pair IgFLNa20–21. The IgFLNa22–23 construct was much more elongated, which is indicative for two independently folded noninteracting domains. Further, the atomic structures solved with NMR spectroscopy show that IgFLNa18–19 forms a pair similar to IgFLNa20–21, but the details of the interaction and orientation of the domains differ. On the other hand, IgFLNa16–17 forms an entirely novel type of domain pair. Sequence comparisons predict that these three interdependent domain pairs are conserved from nematodes to vertebrates, suggesting that the arrangement has special regulatory functions.     

Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20–21

Filamins (FLNs) are large, multidomain actin cross-linking proteins with diverse functions. Besides regulating the actin cytoskeleton, they serve as important links between the extracellular matrix and the cytoskeleton by binding cell surface receptors, functioning as scaffolds for signaling proteins, and binding several other cytoskeletal proteins that regulate cell adhesion dynamics. Structurally, FLNs are formed of an amino terminal actin-binding domain followed by 24 immunoglobulin-like domains (IgFLNs). Recent studies have demonstrated that myosin-mediated contractile forces can reveal hidden protein binding sites in the domain pairs IgFLNa18–19 and 20–21, enabling FLNs to transduce mechanical signals in cells. The atomic structures of these mechanosensor domain pairs in the resting state are known, as well as the structures of individual IgFLN21 with ligand peptides. However, little experimental data is available on how interacting protein binding deforms the domain pair structures. Here, using small-angle x-ray scattering-based modelling, x-ray crystallography, and NMR, we show that the adaptor protein migfilin-derived peptide-bound structure of IgFLNa20–21 is flexible and adopts distinctive conformations depending on the presence or absence of the interacting peptide. The conformational changes reported here may be common for all peptides and may play a role in the mechanosensor function of the site.     

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 β-strand of domain 19, FLNa(19), and the first β-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 β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.     

Migfilin, a Molecular Switch in Regulation of Integrin Activation

The linkage of heterodimeric (α/β) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin β CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.Cells reside in a protein network, the extracellular matrix (ECM).⁴ Cell-ECM contact is crucial for many physiological and pathophysiological processes and is primarily mediated by heterodimeric (α/β) transmembrane receptors, the integrins (1). Integrins engage a variety of ECM proteins via their extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (CTs). The ability of integrins to bind to their ligands is uniquely controlled by the integrin CTs via a process called “inside-out signaling,” i.e. upon cellular stimulation, an integrin, typically expressed in a latent state, can receive intracellular signal(s) at its CT, which transmits through the transmembrane domain to the extracellular domain thereby converting the receptor from a low to a high affinity state (integrin activation). How such long range information transfer is initiated and regulated has been the central topic of integrin/cell adhesion research over the decades (for reviews see Refs. 2-5). Structural/biochemical studies have indicated that the inside-out signaling involves the unclasping of the integrin α/β CT complex (6-9), followed by extensive rearrangement of transmembrane domain and extracellular domain (10-13). Talin, a large actin-linking protein, was found to play a key role in the unclasping process by binding to the integrin β CTs (7-8, 14). Talin activity appears to be controlled by multiple factors or pathways (15-20).Relevant to this study is the role of filamin, another major actin cross-linking protein (21-22), in integrin activation. Filamin was found to share an overlapping binding site on integrin β CTs with talin and thus suppress the talin-integrin interaction (16). Gene silencing of filamin in various cell lines to remove the filamin-integrin connection enhances integrin activation (16, 23), whereas increased filamin-integrin interaction inhibits cell migration (24), a process critically dependent on integrin activation. Together these observations support the notion that filamin binding to integrin serves as a cellular brake to control the dynamics of the integrin activation by inhibiting talin function and ECM-cytoskeleton communication. The mechanism as to how the filamin brake is turned off to promote integrin activation and cell migration is not understood.Filamin is known to contain an N-terminal actin binding domain (ABD) and a long rod-like domain of 24 immunoglobulin-like repeats, of which repeat 21 (IgFLN21) was shown to play a key role in binding to integrin β CTs and blocking the talin-integrin β CT interaction (16). Interestingly, IgFLN21 also recognizes another intracellular protein called migfilin, which has been shown to be an important regulator of integrin-mediated cytoskeletal rearrangements, cell shape change (25), and cell migration (26).In an effort to dissect the complex intermolecular interactions between migfilin, filamin, and integrin, we have undertaken a detailed structural/functional analysis. Using NMR spectroscopy, we have mapped the precise IgFLN21 binding region in migfilin, which is located at the extreme N terminus (residues 1-24) of migfilin (migfilin-N), and we solved solution structure of the IgFLN21-migfilin-N complex. To our surprise, despite little sequence homology, migfilin binds to the same region in IgFLN21 where integrin β CT binds. Detailed NMR and biochemical analyses demonstrate that the migfilin-filamin interaction is an order of magnitude higher than the integrin-filamin interaction and that the migfilin binding to filamin can competitively dissociate filamin from integrin and thus promote the talin-integrin interaction. Using multiple functional approaches, we further show that migfilin, but not its filamin binding defective mutant, significantly enhances integrin activation. These data suggest a novel regulatory pathway in which the binding of filamin to its downstream target migfilin switches off the integrin-filamin connection, thereby promoting talin binding to and activation of integrins.     

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.     

The evolution of filamin - A protein domain repeat perspective

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates.     

Integrin binding immunoglobulin type filamin domains have variable stability

Filamin, a large modular protein composed mainly of many immunoglobulin-like domains, is a potent cross-linker of actin filaments. The region containing immunoglobulin type modules 19-21 makes up the binding site for the cytoplasmic tails of the integrin adhesion receptors. Here we investigate the stability of the Ig-like filamin domains using NMR studies over a range of pH and temperature. We show that the 21st Ig-like module (FLNa21) is partly unfolded even under physiological conditions and when attached to FLNa20. It is, however, appreciably stabilized upon binding to integrins. FLNa21 is noticeably less stable than neighboring homologous modules, such as FLNa19 and FLNa17. This variability in stability could be related to the known sensitivity of filamin to cell-mediated mechanical forces.     

Evidence for multisite ligand binding and stretching of filamin by integrin and migfilin

Filamin, a large cytoskeletal adaptor, connects plasma membrane to cytoskeleton by binding to transmembrane receptor integrin and actin. Seven of 24 filamin immunoglobulin repeats have conserved integrin binding sites, of which repeats 19 and 21 were shown to be autoinhibited by their adjacent repeats 18 and 20, respectively. Here we show using nuclear magnetic resonance spectroscopy that the autoinhibition can be relieved by integrin or integrin regulator migfilin. We further demonstrate that repeats 19 and 21 can simultaneously engage ligands. The data suggest that filamin is mechanically stretched by integrin or migfilin via a multisite binding mechanism for regulating cytoskeleton and integrin-mediated cell adhesion.     

The Regulation Mechanism for the Auto-Inhibition of Binding of Human Filamin A to Integrin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Human filamins are large actin cross-linking proteins that connect integrins to the cytoskeleton. Filamin binding to the cytoplasmic tail of β integrins has been shown to prevent integrin activation in cells, which is important for controlling cell adhesion and migration. The molecular-level mechanism for filamin binding to integrin has been unclear, however, as it was recently demonstrated that filamin undergoes intramolecular auto-inhibition of integrin binding. In this study, using steered molecular dynamics simulations, we found that mechanical force applied to filamin can expose cryptic integrin binding sites. The forces required for this are considerably lower than those for filamin immunoglobulin domain unfolding. The mechanical-force-induced unfolding of filamin and exposure of integrin binding sites occur through stable intermediates where integrin binding is possible. Accordingly, our results support filamin's role as a mechanotransducer, since force-induced conformational changes allow binding of integrin and other transmembrane and intracellular proteins. This observed force-induced conformational change can also be one of possible mechanisms involved in the regulation of integrin activation.     

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.     

Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa·partner complexes as templates, we generated in silico models of IgFLNa·CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated (∼160 nm) strands, whereas CFTR is compact (6∼8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa·partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.     

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K_a ~ 10⁵), the binding of the intact protein to F-actin is strong (apparent K_a ~ 10⁸), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat–F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.     

Filamin A mediates interactions between cytoskeletal proteins that control cell adhesion

Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion receptors, including the β1 integrin. Filamin A is a large, multi-domain, homodimeric actin binding protein that contributes to the mechanical stability of cells and interacts with several proteins that regulate cell adhesion including β1 integrin and several protein kinases. Here we review current data on the structure, mechanical properties and intracellular signaling functions of filamin that regulate cell adhesion. We also consider new data showing that interactions of filamin A with intermediate filaments and protein kinase C enable tight regulation of β1 integrin function and consequently early events in cell adhesion and migration on extracellular matrix proteins.     

Identification and Characterization of Multiple Similar Ligand-binding Repeats in Filamin: IMPLICATION ON FILAMIN-MEDIATED RECEPTOR CLUSTERING AND CROSS-TALK*

The actin-binding protein filamin links membrane receptors to the underlying cytoskeleton. The cytoplasmic domains of these membrane receptors have been shown to bind to various filamin immunoglobulin repeats. Notably, among 24 human filamin repeats, repeat 17 was reported to specifically bind to platelet receptor glycoprotein Ibα and repeat 21 to integrins. However, a complete sequence alignment of all 24 human filamin repeats reveals that repeats 17 and 21 actually belong to a distinct filamin repeat subgroup (containing repeats 4, 9, 12, 17, 19, 21, and 23) that shares a conserved ligand-binding site. Using isothermal calorimetry and NMR analyses, we show that all repeats in this subgroup can actually bind glycoprotein Ibα, integrins, and a cytoskeleton regulator migfilin in similar manners. These data provide a new view on the ligand specificity of the filamin repeats. They also suggest a multiple ligand binding mechanism where similar repeats within a filamin monomer may promote receptor clustering or receptor cross-talking for regulation of the cytoskeleton organization and diverse filamin-mediated cellular activities.     

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.     

Phosphorylation Facilitates the Integrin Binding of Filamin under Force

Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19-21 of filamin-A (IgFLNa-R19-R21). IgFLNa-R19-21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser²¹⁵², however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 β-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser²¹⁵² without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.     

Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation

BackgroundCytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of β subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated.MethodsBinding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking.ResultsThe αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions.ConclusionOur findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state.General significanceFilamin binding to both α and β CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.