An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability.
The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.
Goley
Erin D
ED
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Rammohan
Aravind
A
Znameroski
Elizabeth A
EA
Firat-Karalar
Elif Nur
EN
Sept
David
D
Welch
Matthew D
MD
D008841:Actin Cytoskeleton / Q000378:metabolism*; D051377:Actin-Related Protein 2 / Q000737:chemistry* / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000737:chemistry* / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins
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