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

Daniel V. Iwamoto, Andrew Huehn, Bertrand Simon, Clotilde Huet-Calderwood, Massimiliano Baldassarre, Charles V. Sindelar (Corresponding Author), David A. Calderwood (Corresponding Author)

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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.
Original languageEnglish
Pages (from-to)918-927
Number of pages10
JournalNature Structural & Molecular Biology
Volume25
Early online date17 Sept 2018
DOIs
Publication statusPublished - Oct 2018

Bibliographical note

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.



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.)).

Keywords

  • actin
  • cryoelectron microscope

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