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

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.)).Peer reviewedPostprin

Filamins Regulate Cell Spreading and Initiation of Cell Migration

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration

NMR Structure, Dynamics and Interactions of the Integrin β2 Cytoplasmic Tail with Filamin Domain IgFLNa21

Abstract Integrins are transmembrane proteins that mediate cell adhesion and migration. Each integrin is a heterodimer formed by an α and a β subunit. A large number of cytoplasmic proteins interact with the cytoplasmic tails (CTs) of integrins. The actin-binding cytoskeletal protein filamin A is a negative regulator of integrin activation. The IgFLNa21 domain of filamin A binds to the C-terminus of β2 CT that contains a TTT-motif. Based on x-ray crystallography, it has been reported that the integrin β2 CT forms a β strand that docks into the β strands C and D of IgFLNa21. In this study, we performed solution NMR analyses of IgFLNa21 in the presence of integrin β2 CT peptides, and hybrid IgFLNa21, a construct of covalently linked IgFLNa21 and β2 CT. The atomic resolution structure of the hybrid IgFLNa21 demonstrated conserved binding mode with β2 CT. Although, 15N relaxation, model free analyses and H-D exchange studies have uncovered important insights into the conformational dynamics and stability of β2 CT in complex with IgFLNa21. Such dynamical characteristics are likely to be necessary for the TTT-motif to serve as a phosphorylation switch that regulates filamin A binding to integrin β2 CT

Inter-domain interactions in filamins

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the Nterminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation.peerReviewe