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Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex

Nature Cell Biology volume 3, pages 76–82 (2001)Cite this article

Abstract

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 μM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable ΔH of −10 kcal mol−1 drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 μM; 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.

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Figure 1: WASP constructs and nucleation reactions.
Figure 2: Kinetics and thermodynamics of binding of the WA domain to actin monomers and to the Arp2/3 complex.
Figure 3: Actin-filament nucleation by the Arp2/3 complex activated by WASP-WA constructs.

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Acknowledgements

We thank L. Blanchoin, K. Amann, W-L. Lee (analytical ultracentrifugation), S. C. Koerber (circular-dichroism spectroscopy) and J. Meisenhelder of the Salk Institute for help with this work. This work was supported by NIH Research Grant GM-26338 (to T.D.P.), an NIH Postdoctoral Fellowship (to H.N.H.), a postdoctoral fellowship from Association pour la Recherche sur le Cancer (ARC) and the Philippe Foundation (to J.B.M.), and a gift to the laboratory from S. Hammerslag.

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Authors and Affiliations

  1. Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037, California, USA

    Jean-Baptiste Marchand, Donald A. Kaiser, Thomas D. Pollard & Henry N. Higgs

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  1. Jean-Baptiste Marchand
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  2. Donald A. Kaiser
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Correspondence to Thomas D. Pollard.

Supplementary information

Figure S1

Mutations in WASP-WA do not affect actin-filament branching or interaction with WASP-GBD. (PDF 381 kb)

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Marchand, JB., Kaiser, D., Pollard, T. et al. Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat Cell Biol 3, 76–82 (2001). https://doi.org/10.1038/35050590

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