Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the Arp2/3 complex.
The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.
Kelly
Alexander E
AE
Graduate Group in Biophysics and Department of Cellular & Molecular Pharmacology, University of California-San Francisco, San Francisco, CA 94143, USA.
Kranitz
Heather
H
Dötsch
Volker
V
Mullins
R Dyche
RD
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism*; D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D016880:Anisotropy; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism / Q000502:physiology*; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000378:metabolism / Q000502:physiology*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D003432:Cross-Linking Reagents; D010455:Peptides; C114864:WASF1 protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family; D005982:Glutathione Transferase