Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis of the filamin A actin-binding domain interaction with F-actin

Nature Structural & Molecular Biology volume 25, pages 918–927 (2018)Cite this article

Subjects

Abstract

Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-cross-linking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first calponin homology domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD–F-actin binding.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cryo-EM map and model of FLNaABD-E254K bound to F-actin.
Fig. 2: ABS-N contributes to F-actin binding.
Fig. 3: ABS2 and ABS2’ facilitate major binding interactions with F-actin.
Fig. 4: Opening of the ABD is required to avoid steric clashes and facilitate actin binding.
Fig. 5: ABD opening is mediated by an inter-CH domain latch.
Fig. 6: FLNaCH1 domain mutations confer a loss of function to F-actin binding.

Data availability

Cryo-EM reconstructions were deposited in the Electron Microscopy Data Bank with the following accession numbers: F20-F-actin-FLNaABD, EMD-7833; F20-F-actin-FLNaABD-Q170P, EMD-7832; F20-F-actin-FLNaABD-E254K, EMD-8918; Krios-F-actin-FLNaABD-E254K, EMD-7831. The corresponding FLNaABD-E254K filament model was deposited in the PDB with accession number 6D8C. Source data for F-actin-targeting analyses (Figs. 2c,d,g,h, 3b,c,e,f, 4d,e, 5c,d, and 6a,b) and co-sedimentation assays (Figs. 5g and 6d) are available with the paper online. Other data are available from the corresponding author upon reasonable request.

References

  1. Pollard, T. D. Actin and actin-binding proteins. Cold Spring Harb. Perspect. Biol. 8, 575–591 (2016).

    Article  CAS  Google Scholar 

  2. Svitkina, T. The actin cytoskeleton and actin-based motility. Cold Spring Harb. Perspect. Biol. 10, 1–22 (2018).

    Article  Google Scholar 

  3. Robertson, S. P. Filamin A: phenotypic diversity. Curr. Opin. Genet. Dev. 15, 301–307 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Sjöblom, B., Ylänne, J. & Djinović-Carugo, K. Novel structural insights into F-actin-binding and novel functions of calponin homology domains. Curr. Opin. Struct. Biol. 18, 702–708 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Gimona, M., Djinovic-Carugo, K., Kranewitter, W. J. & Winder, S. J. Functional plasticity of CH domains. FEBS Lett. 513, 98–106 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Ruskamo, S. & Ylänne, J. Structure of the human filamin A actin-binding domain. Acta Crystallogr. D Struct. Biol. 65, 1217–1221 (2009).

    Article  CAS  Google Scholar 

  7. Clark, A. R., Sawyer, G. M., Robertson, S. P. & Sutherland-Smith, A. J. Skeletal dysplasias due to filamin A mutations result from a gain-of-function mechanism distinct from allelic neurological disorders. Hum. Mol. Genet. 18, 4791–4800 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Bresnick, A. R., Warren, V. & Condeelis, J. Identification of a short sequence essential for actin binding by dictyostelium ABP-120. J. Biol. Chem. 265, 9236–9240 (1990).

    CAS  PubMed  Google Scholar 

  9. Levine, B. A., Moir, A. J. G., Patchell, V. B. & Perry, S. V. The interaction of actin with dystrophin. FEBS Lett. 263, 159–162 (1990).

    Article  CAS  PubMed  Google Scholar 

  10. Karinch, A. M., Zimmer, W. E. & Goodman, S. R. The identification and sequence of the actin-binding domain of human red blood cell beta spectrin. J. Biol. Chem. 265, 11833–11840 (1990).

    CAS  PubMed  Google Scholar 

  11. Levine, B., Moir, A., Patchell, V. & Perry, S. Binding sites involved in the interaction of actin with the N-terminal region of dystrophin. FEBS Lett. 298, 44–48 (1992).

    Article  CAS  PubMed  Google Scholar 

  12. Hemmings, L., Kuhlman, P. A. & Critchley, D. R. Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain. J. Cell. Biol. 116, 1369–1380 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Corrado, K., Mills, P. & Chamberlain, J. Deletion analysis of the dystrophin-actin binding domain. FEBS Lett. 344, 255–260 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Goldsmith, S. C. et al. The structure of an actin-crosslinking domain from human fimbrin. Nat. Struct. Biol. 4, 708–712 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Keep, N. H. et al. Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer. Structure 7, 1539–1546 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Norwood, F. L. M., Sutherland-Smith, A. J., Keep, N. H. & Kendrick-Jones, J. The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy. Structure 8, 481–491 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Liu, J., Taylor, D. W. & Taylor, K. A. A 3-D reconstruction of smooth muscle α-actinin by CryoEm reveals two different conformations at the actin-binding region. J. Mol. Biol. 338, 115–125 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Galkin, V. E., Orlova, A., Cherepanova, O., Lebart, M.-C. & Egelman, E. H. High-resolution cryo-EM structure of the F-actin-fimbrin/plastin ABD2 complex. Proc. Natl Acad. Sci. USA 105, 1494–1498 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Galkin, V. E., Orlova, A., Salmazo, A., Djinovic-Carugo, K. & Egelman, E. H. Opening of tandem calponin homology domains regulates their affinity for F-actin. Nat. Struct. Mol. Biol. 17, 614–616 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lin, A. Y., Prochniewicz, E., James, Z. M., Svensson, B. & Thomas, D. D. Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism. Proc. Natl Acad. Sci. USA 108, 12729–12733 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Broderick, M. J. F., Bobkov, A. & Winder, S. J. Utrophin ABD binds to F-actin in an open conformation. FEBS Open Bio 2, 6–11 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Avery, A. W. et al. Structural basis for high-affinity actin binding revealed by a beta-III-spectrin SCA5 missense mutation. Nat. Commun. 8, 1–7 (2017).

    Article  CAS  Google Scholar 

  23. Nakamura, F., Osborn, T. M., Hartemink, Ca, Hartwig, J. H. & Stossel, T. P. Structural basis of filamin A functions. J. Cell Biol. 179, 1011–1025 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Razinia, Z., Mäkelä, T., Ylänne, J. & Calderwood, D. A. Filamins in mechanosensing and signaling. Annu. Rev. Biophys. 41, 227–246 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nakamura, F., Stossel, T. P. & Hartwig, J. H. The filamins: organizers of cell structure and function. Cell Adh. Migr. 5, 160–169 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Sheen, V. L. et al. Mutations in the X-linked filamin 1 gene cause periventricular nodular heterotopia in males as well as in females. Hum. Mol. Genet. 10, 1775–1783 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Parrini, E. et al. Periventricular heterotopia: phenotypic heterogeneity and correlation with Filamin A mutations. Brain 72, 1892–1906 (2006).

    Article  Google Scholar 

  28. Chen, M. H. et al. Thoracic aortic aneurysm in patients with loss of function Filamin A mutations: clinical characterization, genetics, and recommendations. Am. J. Med. Genet. 176A, 337–350 (2018).

    Article  CAS  Google Scholar 

  29. Solé, G. et al. Bilateral periventricular nodular heterotopia in France: frequency of mutations in FLNA, phenotypic heterogeneity and spectrum of mutations. J. Neurol. Neurosurg. Psychiatry 80, 1394–1398 (2009).

    Article  PubMed  Google Scholar 

  30. Robertson, S. P. Otopalatodigital syndrome spectrum disorders: otopalatodigital syndrome types 1 and 2, frontometaphyseal dysplasia and Melnick–Needles syndrome. Eur. J. Hum. Genet. 15, 3–9 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Robertson, S. P. et al. Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans. Nat. Genet. 33, 487–491 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Mentes, A. et al. High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing. PNAS 115, 1292–1297 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Razinia, Z., Baldassarre, M., Cantelli, G. & Calderwood, D. A. ASB2α, an E3 ubiquitin ligase specificity subunit, regulates cell spreading and triggers proteasomal degradation of filamins by targeting the filamin calponin homology 1 domain. J. Biol. Chem. 288, 32093–32105 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Singh, S. M., Bandi, S. & Mallela, K. M. G. The N-terminal flanking region modulates the actin binding affinity of the utrophin tandem calponin-homology domain. Biochemistry 56, 2627–2636 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Weins, A. et al. Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity. Proc. Natl Acad. Sci. USA 104, 16080–16085 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Valdés-Mas, R. et al. Mutations in filamin C cause a new form of familial hypertrophic cardiomyopathy. Nat. Commun. 5, 5326 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Bagnall, R. D., Molloy, L. K., Kalman, J. M. & Semsarian, C. Exome sequencing identifies a mutation in the ACTN2 gene in a family with idiopathic ventricular fibrillation, left ventricular noncompaction, and sudden death. BMC Med. Genet. 15, 99 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kunishima, S. et al. ACTN1 mutations cause congenital macrothrombocytopenia. Am. J. Hum. Genet. 92, 431–438 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Theis, J. L. et al. Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochem. Biophys. Res. Commun. 351, 896–902 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Jenkins, Z. A. et al. Differential regulation of two FLNA transcripts explains some of the phenotypic heterogeneity in the loss-of-function filaminopathies. Hum. Mutat. 39, 103–113 (2018).

    Article  CAS  PubMed  Google Scholar 

  41. Sawyer, G. M., Clark, A. R., Robertson, S. P. & Sutherland-Smith, A. J. Disease-associated substitutions in the filamin B actin binding domain confer enhanced actin binding affinity in the absence of major structural disturbance: insights from the crystal structures of filamin B actin binding domains. J. Mol. Biol. 390, 1030–1047 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Kostan, J., Gregor, M., Walko, G. & Wiche, G. Plectin isoform-dependent regulation of keratin-integrin α6β4 anchorage via Ca2+/calmodulin. J. Biol. Chem. 284, 18525–18536 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Song, J. G. et al. Structural insights into Ca2+-calmodulin regulation of plectin 1a-integrin β4 interaction in hemidesmosomes. Structure 23, 558–570 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nakamura, F., Hartwig, J. H., Stossel, T. P. & Szymanski, P. T. Ca2+ and calmodulin regulate the binding of filamin A to actin filaments. J. Biol. Chem. 280, 32426–32433 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. García-Alvarez, B., Bobkov, A., Sonnenberg, A. & De Pereda, J. M. Structural and functional analysis of the actin binding domain of plectin suggests alternative mechanisms for binding to F-actin and integrin β4. Structure 11, 615–625 (2003).

    Article  PubMed  Google Scholar 

  46. Kiema, T. et al. The molecular basis of filamin binding to integrins and competition with talin. Mol. Cell 21, 337–347 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Heuzé, M. L. et al. ASB2 targets filamins A and B to proteasomal degradation. Blood 112, 5130–5140 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lad, Y. et al. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding. EMBO J. 26, 3993–4004 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Razinia, Z. et al. The E3 ubiquitin ligase specificity subunit ASB2α targets filamins for proteasomal degradation by interacting with the filamin actin-binding domain. J. Cell. Sci. 124, 2631–2641 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using µmanager. Curr. Protoc. Mol. Biol. 92, 14.20.1–14.20.17 (2010).

    Google Scholar 

  51. Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. MacGrath, S. M. & Koleske, A. J. Arg/Abl2 modulates the affinity and stoichiometry of binding of cortactin to F-actin. Biochemistry 51, 6644–6653 (2012).

    Article  CAS  PubMed  Google Scholar 

  53. Heier, J. A., Dickinson, D. J. & Kwiatkowski, A. V. Measuring protein binding to F-actin by co-sedimentation. J. Vis. Exp. 123, 1–8 (2017).

    Google Scholar 

  54. Boivin, S., Kozak, S. & Meijers, R. Optimization of protein purification and characterization using Thermofluor screens. Protein Expr. Purif. 91, 192–206 (2013).

    Article  CAS  PubMed  Google Scholar 

  55. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Grant, T. & Grigorieff, N. Automatic estimation and correction of anisotropic magnification distortion in electron microscopes. J. Struct. Biol. 192, 204–208 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  PubMed  Google Scholar 

  59. Egelman, E. H. Reconstruction of helical filaments and tubes. Methods Enzymol. 482, 167–183 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Egelman, E. H. A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85, 225–234 (2000).

    Article  CAS  PubMed  Google Scholar 

  61. He, S. & Scheres, S. H. W. Helical reconstruction in RELION. J. Struct. Biol. 198, 163–176 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Huehn, A. et al. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments. J. Biol. Chem. 293, 5377–5383 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).

    Article  PubMed  Google Scholar 

  65. Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  66. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Struct. Biol. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  67. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Struct. Biol. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  68. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Struct. Biol. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  69. Hastings, J. et al. ChEBI in 2016: improved services and an expanding collection of metabolites. Nucleic Acids Res. 44, D1214–D1219 (2016).

    Article  CAS  PubMed  Google Scholar 

  70. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Electronic ligand builder and optimization workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Struct. Biol. 65, 1074–1080 (2009).

    Article  CAS  Google Scholar 

  71. Gallivan, J. P. & Dougherty, D. A. Cation-pi interactions in structural biology. Proc. Natl Acad. Sci. USA 96, 9459–9464 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Z. Razinia for generating numerous FLNa constructs, S. Wu for expertise in using the Krios microscope, J. Lees for advice on model refinement, and M. Lemmon for helpful comments in preparing the manuscript. We also thank the Yale Center for Research Computing for guidance and use of the Farnam Cluster, as well as the staff at the YMS Center for Molecular Imaging for the use of the EM Core Facility. This work was funded by grants from the National Institutes of Health (R01-GM068600 (D.A.C.), R01-NS093704 (D.A.C.), R37-GM057247 (C.V.S.), R01-GM110530 (C.V.S.), T32-GM007324, T32-GM008283) and an award from American Heart Association (15PRE25700119 (D.V.I.)).

Author information

Author notes

  1. These authors contributed equally: D. V. Iwamoto, A. Huehn.

Authors and Affiliations

  1. Department of Pharmacology, Yale University, New Haven, CT, USA

    Daniel V. Iwamoto, Bertrand Simon, Clotilde Huet-Calderwood, Massimiliano Baldassarre & David A. Calderwood

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Andrew Huehn & Charles V. Sindelar

  3. Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK

    Massimiliano Baldassarre

  4. Department of Cell Biology, Yale University, New Haven, CT, USA

    David A. Calderwood

Authors
  1. Daniel V. Iwamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew Huehn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bertrand Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Clotilde Huet-Calderwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Massimiliano Baldassarre
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles V. Sindelar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David A. Calderwood
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.V.I., M.B., and D.A.C. conceived the project. D.V.I., B.S., C.H.C., and M.B. designed constructs and collected and interpreted biochemical and cellular data. A.H. and D.V.I. prepared cryo-EM samples and collected cryo-EM data. A.H. and C.V.S. performed cryo-EM analysis and model refinement. D.V.I., A.H., C.V.S., and D.A.C. wrote the manuscript with contributions from all other authors.

Corresponding authors

Correspondence to Charles V. Sindelar or David A. Calderwood.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 FLNaABD–F-actin complexes.

a, High-frequency noise-substituted Fourier shell correlation calculations between independently refined half maps reveals the resolution of each map. The FSC curve between the final FLNaABD-E254K–F-actin map and refined model is shown in purple. b, A 10-Å-filtered cryo-EM density map of wild-type (WT) FLNaABD (left and middle) was rigid-body docked with crystal structures for actin (PDB 6C1D; different subunits in dark blue, cyan, and light blue) and FLNaCH1 (PDB 3HOP; green) (right). c, A 6.6-Å-filtered cryo-EM density map of the gain-of-function mutant FLNaABD-Q170P (left and middle) was docked with the refined actin and FLNaCH1 models (right) and colored as in b. d, A 7.4-Å-filtered cryo-EM density map of the gain-of-function mutant FLNaABD-E254K (left and middle) was docked with the refined actin and FLNaCH1 models (right) and colored as in b. Observed extra density for FLNaCH2 is shown in tan.

Supplementary Figure 2 ABS-N mutations inhibit FLNaABD–F-actin binding.

a, Micrographs of mouse NIH-3T3 fibroblasts transiently transfected with GFP or FLNaABD-GFP ± N-terminal truncation constructs after fixation, permeabilization, and staining with Alexa Fluor 568–phalloidin to visualize F-actin filaments. Cells were imaged in the green (left; GFP signal) and red (right; F-actin signal) channels. Scale bar, 20 µm. WT, wild type. b, Anti-GFP immunoblot on lysates from NIH-3T3 cells transiently transfected with GFP or FLNaABD-GFP ± N-terminal truncations. Vinculin was used as a loading control. c, Micrographs of fibroblasts transfected with GFP or FLNaABD-GFP constructs containing ABS-N mutations, prepared and imaged as in a. Scale bar, 20 µm. d, Anti-GFP immunoblot on lysates from NIH-3T3 cells transiently transfected with GFP or FLNaABD-GFP ± ABS-N mutations. Vinculin was used as a loading control.

Supplementary Figure 3 Immunoblotting of ABS2ʹ and ABS2 mutant FLNaABDs.

a, Anti-GFP immunoblot on lysates from NIH-3T3 cells transiently transfected with GFP or FLNaABD-GFP ± ABS2ʹ mutations. Vinculin was used as a loading control. WT, wild type. b, Anti-GFP immunoblot on lysates from NIH-3T3 cells transiently transfected with GFP or FLNaABD-GFP ± ABS2 mutations. Vertical separations between panels indicate the exclusion of non-relevant lane samples from the same gel. Vinculin was used as a loading control.

Supplementary Figure 4 OPDSD-associated mutations increase FLNaABD binding to F-actin.

a, Micrographs of mouse NIH-3T3 fibroblasts transiently transfected with GFP or FLNaABD-GFP ± the Q170P or E254K mutation after fixation, permeabilization, and staining with Alexa Fluor 568–phalloidin to visualize actin filaments. Cells were imaged in the green (left; GFP signal) and red (right; F-actin signal) channels. Scale bar, 20 µm. WT, wild type. b, Top and bottom, anti-GFP immunoblots on lysates from NIH-3T3 cells transiently transfected with GFP, FLNaABD-GFP or isolated FLNaCH-GFP domains ± the Q170P or E254K mutation. Vinculin was used as a loading control. Vertical separations between panels indicate the exclusion of non-relevant lane samples from the same gel. c, Anti-GFP immunoblot on lysates from NIH-3T3 cells transiently transfected with GFP, FLNaABD-GFP or FLNaABD-W142A-GFP. Vinculin was used as a loading control. d, Table summarizing apparent dissociation constants (Kd, µM) and Bmax (molar ratio ABD:actin) from co-sedimentation assays with purified wild-type or mutant 6 × His-FLNaABD proteins, ± s.d.

Supplementary Figure 5 Immunoblotting and size-exclusion chromatography of FLNaABD with PVNH-associated mutations.

a, Top and bottom, anti-GFP immunoblots on lysates from NIH-3T3 cells transiently transfected with GFP or FLNaABD-GFP ± PVNH mutations. Vinculin was used as a loading control. WT, wild type. b, Analytical size-exclusion chromatography of purified bacterially expressed 6 × His-FLNaABD-WT, 6 × His-FLNaABD-A39G, or 6 × His-FLNaABD-A128V. Protein concentration was measured by UV absorbance at 280 nm (arbitrary units, mAU). c, Top, the FLNa residue A128 is located in helix F of the CH1 domain and does not contact actin, although its mutation to valine would likely perturb this short helix (see space-filling models, bottom) which contains important actin-binding residues (not shown) and results in a loss of actin binding. d, The FLNaCH1 residues E82 and S149 (mutated in PVNH) are situated far from F-actin-binding sites in the actin-bound cryo-EM structure.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Note

Reporting Summary

Supplementary Dataset 1

Uncropped Coomassie-stained SDS–PAGE gels from co-sedimentation assays

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Iwamoto, D.V., Huehn, A., Simon, B. et al. Structural basis of the filamin A actin-binding domain interaction with F-actin. Nat Struct Mol Biol 25, 918–927 (2018). https://doi.org/10.1038/s41594-018-0128-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-018-0128-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing