Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Department of Physiology and Biophysics, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 10/2012; 424(5). DOI: 10.1016/j.jmb.2012.09.025
Source: PubMed


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 interaction appears to be specific, since no other fragment of the FLNa molecule or individual Ig repeats examined, besides ones with 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.

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    • "The Ig repeats are further segmented by two calpain-sensitive hinges, resulting in rod-1 (repeats 1e15), rod-2 (repeats 16e23), and dimerization domain (repeat 24) [2]. FLNA protein crosslinks actin filaments through the N-terminal actin-binding domain and rod-1 segment, whereas rod- 2 segment is free from F-actin [3] [4]. FLNA connects F-actin to membrane proteins such as integrin and glycoproteins to stabilize plasma membrane and to transmit signals [1,5e7]. "
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