Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex.
The Wiskott-Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol(-1) drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.
Marchand
J B
JB
Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Kaiser
D A
DA
Pollard
T D
TD
Higgs
H N
HN
D008841:Actin Cytoskeleton / Q000378:metabolism; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D005454:Fluorescence Polarization / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D017354:Point Mutation / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012235:Rhodamines; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D011506:Proteins; D012235:Rhodamines; C495767:WAS protein, human; C114864:WASF1 protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family