The structure explains the inhibited nature of the WRC. In the complex, the WAVE1 VCA is bound by a conserved surface of Sra1 and residues 82-184 of WAVE1, which form five helices (α2-α6) and a series of intervening loops. This element of WAVE1 traces a meandering path across a concave surface of Sra1, and we refer to it as the “meander region” ( and Supplementary Fig. 8
). Contacts between the meander region and Sra1 bury over 2,100 Å2
(~56% of the total WAVE1-Sra1 interface; Supplementary Fig. 9
). The meander sequence is highly conserved among the different WAVE proteins (Supplementary Fig. 8
), as is its contact surface on Sra1, suggesting its interactions and irregular structure are conserved.
The V- and C-regions of the VCA lie on the surface of Sra1 and form two amphipathic helices (residues 500-514 and 531-543, respectively) that also pack against α2 and α6 of WAVE1, respectively (Supplementary Fig. 10
). The A-region of the VCA (residues 545-559) is likely disordered, as it is not observed in the electron density. During actin filament nucleation, the V-region recruits an initial actin monomer to the nascent filament, while the C- and A-regions contribute binding energy and induce activating conformational changes in the Arp2/3 complex26,27
. The structure and complementary experimental data below indicate that sequestration of both the V- and the C-regions by Sra1 and the meander region of WAVE1 underlies VCA inhibition within the WRC.
Inhibition of the V-region involves a combination of contacts to actin-binding residues and induction of structure that is incompatible with actin binding (). In the complex of the V-region of WAVE2 with actin28
, residues equivalent to 497-507 of WAVE1 form a helix that inserts into the cleft between actin subdomains 1 and 3. Residues equivalent to 508-516 are extended, and the Ile509 and Arg512 equivalents contact actin. In the structure of miniWRC, the entire V-region is helical, and the side chains of Leu 501, Leu 502, Ile 505, Ile 509 and Arg 512 are buried in the Sra1 interface, making the V-region inaccessible to actin. Sequestration of the V-region is an important contributor to WRC inhibition, as mutating V-helix contact residues Leu 841, Phe 844 and Trp 845 of Sra1 constitutively activates miniWRC toward the Arp2/3 complex (L841A/F844A/W845ASra1
-miniWRC, ), producing branched filaments (Supplementary Fig. 11
The C-helix is also critical for activation of Arp2/3 complex29
, as mutations of Val 531, Leu 535 or Arg 538 in the WAVE1 VCA reduce activity toward Arp2/3 complex by at least 50%. In the miniWRC, the C-helix buries its hydrophobic face in the Sra1-α6 interface (, Supplementary Figs. 8 and 9
). Intermolecularly, Val 531, Ala 532, Leu 535 and Ile 539 of WAVE1 make van der Waals contacts with Sra1. Intramolecularly, Val 531, Ile 534, Leu 535, Arg 538 and Val 541 of the C-helix pack against α6. Hence, the structure shows that the WRC sequesters C-helix residues important for activation of the Arp2/3 complex, resulting in inhibition. This mechanism is also supported by mutagenesis: perturbing contacts of the C-helix with either Sra1 (L697D/Y704DSra1
-miniWRC, ) or the α6 helix (W161E/K162DWAVE1
-miniWRC, ) leads to constitutive activation of the miniWRC in vitro
. Additionally, replacement of wild type WAVE2 with equivalent levels of the analogous α6 mutant (W160E/K161DWAVE2
) in HeLa cells does not alter the integrity of the complex (Supplementary Fig. 4
), but causes a dramatic redistribution of actin, with loss of stress fibers and assembly of filaments at the cell periphery, again consistent with constitutive activation of the WRC ().
Mechanisms of WRC activation by Rac1 and phosphorylation
Interactions of the VCA, and perhaps the structure of the entire meander-VCA element, appear to be highly cooperative, as perturbation of either the V- or C-region contacts produces WRC activity near that of the isolated VCA ().