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ABSTRACT: 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.
Journal of the American Chemical Society 03/2012; 134(15):6660-72. · 9.91 Impact Factor
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ABSTRACT: Filamins are essential, evolutionarily conserved, modular, multidomain, actin-binding proteins that organize the actin cytoskeleton and maintain extracellular matrix connections by anchoring actin filaments to transmembrane receptors. By cross-linking and anchoring actin filaments, filamins stabilize the plasma membrane, provide cellular cortical rigidity, and contribute to the mechanical stability of the plasma membrane and the cell cortex. In addition to binding actin, filamins interact with more than 90 other binding partners including intracellular signaling molecules, receptors, ion channels, transcription factors, and cytoskeletal and adhesion proteins. Thus, filamins scaffold a wide range of signaling pathways and are implicated in the regulation of a diverse array of cellular functions including motility, maintenance of cell shape, and differentiation. Here, we review emerging structural and functional evidence that filamins are mechanosensors and/or mechanotransducers playing essential roles in helping cells detect and respond to physical forces in their local environment.
Annual Review of Biophysics 02/2012; 41:227-46. · 13.57 Impact Factor
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ABSTRACT: The Rap1-GTP interacting adapter protein (RIAM) is an important protein in Rap1-mediated integrin activation. By binding to both Rap1 GTPase and talin, RIAM recruits talin to the cell membrane, thus facilitating talin-dependent integrin activation. In this article, we studied the role of the RIAM Ras-association (RA) and pleckstrin-homology (PH) domains in the interaction with Rap1. We found that the RA domain was sufficient for GTP-dependent interaction with Rap1B, and the addition of the PH domain did not change the binding affinity. We also detected GTP-independent interaction of Rap1B with the N-terminus of RIAM. In addition, we found that the PH domain stabilized the RA domain both in vitro and in cells.
PLoS ONE 01/2012; 7(4):e31955. · 4.09 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 06/2011; 286(30):26921-30. · 4.77 Impact Factor
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ABSTRACT: Filamin A (FLNa) is a large dimeric protein that binds to actin filaments via its actin-binding domain (ABD). The crystal structure of this domain was solved at 3.2 A resolution. The domain adopts a closed conformation typical of other ABDs, but also forms a dimer both in crystallization conditions and in solution. The structure shows the localization of the residues mutated in patients with periventricular nodular heterotopia or otopalatodigital syndrome. Structural analysis predicts that mutations in both types of disorder may affect actin binding.
Acta crystallographica. Section D, Biological crystallography 11/2009; 65(Pt 11):1217-21. · 12.67 Impact Factor
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ABSTRACT: 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 beta 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.
Journal of Molecular Biology 09/2009; 393(3):644-57. · 4.00 Impact Factor
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ABSTRACT: Myotilin is a 57 kDa actin-binding and -bundling protein that consists of a unique serine-rich amino-terminus, two Ig-domains and a short carboxy-terminus with a PDZ-binding motif. Myotilin localizes in sarcomeric Z-discs, where it interacts with several sarcomeric proteins. Point mutations in myotilin cause muscle disorders morphologically highlighted by sarcomeric disarray and aggregation. The actin-binding and dimerization propensity of myotilin has been mapped to the Ig-domains. Here we present high-resolution structure of the first Ig-domain of myotilin (MyoIg1) determined with solution state NMR spectroscopy. Nearly complete chemical shift assignments of MyoIg1 were achieved despite several missing backbone 1H-15N-HSQC signals. The structure derived from distance and dihedral angle restraints using torsion angle dynamics was further refined using molecular dynamics. The structure of MyoIg1 exhibits I-type Ig-fold. The absence of several backbone 1H-15N-HSQC signals can be explained by conformational exchange taking place at the hydrophobic core of the protein.
Journal of Biomolecular NMR 07/2009; 44(2):107-12. · 3.61 Impact Factor
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ABSTRACT: Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16-17 and 18-19. Chemical shift mapping between FLNa tandem Ig domain 16-17 and isolated domain 17 suggests a novel domain-domain interaction mode.
Biomolecular NMR Assignments 07/2009; 3(1):53-6. · 0.72 Impact Factor
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Fumihiko Nakamura,
Outi Heikkinen,
Olli T Pentikäinen,
Teresia M Osborn,
Karen E Kasza,
David A Weitz,
Olga Kupiainen,
Perttu Permi,
Ilkka Kilpeläinen, Jari Ylänne,
John H Hartwig,
Thomas P Stossel
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ABSTRACT: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.
We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.
Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.
PLoS ONE 02/2009; 4(3):e4928. · 4.09 Impact Factor
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ABSTRACT: Abstract
Background
The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown.
Results
In this study, using circular dichroism and nuclear magnetic resonance (NMR), we showed that the ALP internal region (residues 107–273) was largely unfolded in solution, but was able to interact with the α-actinin rod domain in vitro , and to co-localize with α-actinin on stress fibres in vivo . NMR analysis revealed that the titration of ALP with the α-actinin rod domain induces stabilization of ALP. A synthetic peptide (residues 175–196) that contained the N-terminal half of the ZM motif was found to interact directly with the α-actinin rod domain in surface plasmon resonance (SPR) measurements. Short deletions at or before the ZM motif abrogated the localization of ALP to actin stress fibres.
Conclusion
The internal region of ALP appeared to be largely unstructured but functional. The ZM motif defined part of the interaction surface between ALP and the α-actinin rod domain.
BMC Cell Biology. 01/2009;
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ABSTRACT: 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 Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21
interaction and migfilin and integrin β 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.
Journal of Biological Chemistry 12/2008; 283(50):35154-35163. · 4.77 Impact Factor
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ABSTRACT: Tandem calponin homology (CH) domains are well-known actin filaments (F-actin) binding motifs. There has been a continuous debate about the details of CH domain-actin interaction, mainly because atomic level structures of F-actin are not available. A recent electron microscopy study has considerably advanced our structural understanding of CH domain:F-actin complex. On the contrary, it has recently also been shown that CH domains can bind other macromolecular systems: two CH domains from separate polypeptides Ncd80, Nuf2 can form a microtubule-binding site, as well as tandem CH domains in the EB1 dimer, while the single C-terminal CH domain of alpha-parvin has been observed to bind to a alpha-helical leucin-aspartate rich motif from paxillin.
Current Opinion in Structural Biology 11/2008; 18(6):702-8. · 9.42 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 10/2008; 283(50):35154-63. · 4.77 Impact Factor
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ABSTRACT: Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.
The EMBO Journal 10/2007; 26(17):3993-4004. · 9.20 Impact Factor
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ABSTRACT: Earlier reports have shown that ALP has an internal interaction site. We were able to stabilize the structure of this unfolded part to a great extent by aspartic acid, which allowed the backbone assignment. No secondary structure of the polypeptide was observed.
Biomolecular NMR Assignments 08/2007; 1(1):85-7. · 0.72 Impact Factor
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ABSTRACT: 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.
Journal of Molecular Biology 06/2007; 368(4):1011-23. · 4.00 Impact Factor
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ABSTRACT: Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.
Experimental Cell Research 06/2006; 312(8):1299-311. · 3.58 Impact Factor
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ABSTRACT: Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.
Blood 04/2006; 107(5):1925-32. · 9.90 Impact Factor
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ABSTRACT: 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.
Molecular Cell 03/2006; 21(3):337-47. · 14.18 Impact Factor
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ABSTRACT: Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.
Structure 02/2005; 13(1):111-9. · 6.35 Impact Factor
