title: Electron microscopy and 3D reconstruction reveals filamin Ig domain binding to F-actin.
abstract: 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)~10(5)), the binding of the intact protein to F-actin is strong (apparent K(a)~10(8)), 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.
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Worawit
authors.initials: W
authors.lastname: Suphamungmee
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Nakamura
authors.forename: John H
authors.initials: JH
authors.lastname: Hartwig
authors.forename: William
authors.initials: W
authors.lastname: Lehman
mesh_terms: D000199:Actins / Q000378:metabolism; D003285:Contractile Proteins / Q000737:chemistry* / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D021621:Imaging, Three-Dimensional; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008854:Microscopy, Electron; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins