Abstract
Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.
Highlights
VCA in Wiskott-Aldrich syndrome proteins (WASP) proteins binds Arp2/3 complex and actin via a variable number of WASP homology 2 (WH2) domains
In a first step toward understanding the general molecular mechanism for filament branching by WASP proteins, in particular the functional role of the association of actin to their WH2 domain(s) in the branching reaction, we describe here the crystal structure of the VC moiety of neural Wiskott-Aldrich syndrome protein (N-WASP) in complex with ATP-G-actin, in the absence of either DNase I, gelsolin, or latrunculin A
The Stokes radii of these complexes are larger than the Stokes radius of actin (30 Å) or of known 1:1 WH21⁄7ATP1⁄7G-actin complexes obtained with Cobl constructs of 8 – 8.9 kDa containing a single WH2 domain (34 –35 Å), indicating that two actin molecules are bound to V, VC, and VCA
Summary
VCA in WASP proteins binds Arp2/3 complex and actin via a variable number of WH2 domains. The structural organization and stoichiometry of the active VCA1⁄7actin1⁄7Arp2/3 complex (called “branching complex”), its mode of binding to a filament, the nature of elementary steps and transients in the branching process, the fate of all protein partners in the branching reaction, and the possible requirement of additional G-actin molecules to nucleate a daughter filament are missing pieces of the puzzle [23,24,25,26,27,28]. Only one of the two WH2 domains of N-WASP binds actin in the crystal structure of VC-actin, in the isolated V, VC, and VCA domains of N-WASP these two WH2 domains form 1:1 (VA) and 1:2 (VA2) complexes with G-actin (A) in solution, which participate in barbed end assembly like profilin-actin Despite their binding two actin molecules, V, VC and VCA do not nucleate nor sever filaments. Binding of the second actin to VCA is abolished in the ternary VCA1⁄7actin1⁄7Arp2/3 complex
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