Structure and control of the actin regulatory WAVE complex

Abstract

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

Figure 1. MiniWRC structure

a, Stereo view of miniWRC. Sra1, Nap1, WAVE1, Abi2 and HSPC300 are green, blue, magenta, orange and yellow, respectively. The A-region (residues 545-559), α6-V region linker (residues 185-485) and sequence connecting V- and C-helices (residues 519-528) are not observed in the electron density. Latter two shown as dashed lines. b. 180° rotation about a horizontal axis from a. Polybasic region and proposed Rac1 and eIF4E binding sites indicated.

Figure 2. Mechanism of WRC inhibition

a, MiniWRC (rotated 90°about a horizontal axis from Fig. 1a). Sra1 and Nap1 are grey surfaces with conserved residues green and cyan, respectively. Ribbons colored as in Fig. 1. Meander region indicated with dashed line. b, V-helix-Sra1 interactions. Hydrogen bonds are dashed. Green dots indicate actin binding residues. c, C-helix binding interface. Green dots indicate residues important for Arp2/3 activation. d, e, Arp2/3-mediated pyrene-actin assembly assays of miniWRC mutants. A.U., arbitrary units. d, L697D/Y704D_Sra1-miniWRC, L841A/F844A/W845A_Sra1-miniWRC, with Sra1 mutated at C- and V-helix binding site, respectively. e, W161E/K162D_WAVE1-miniWRC, with WAVE1 mutated at C-helix contact site.

Figure 3. Mechanisms of WRC activation by Rac1 and phosphorylation

a. HeLa cells were transfected with shVector-YFP control, or vectors simultaneously suppressing WAVE2 and expressing shRNA-resistant YFP-tagged WAVE2s (green) and scored blindly for lamellipodial phenotype. F-actin visualized with phalloidin (red). Error bars in lower right panel show standard deviation for at least three independent measurements. b, Fractional saturation of WRC versus free Rac1-GMPPNP measured by equilibrium dialysis. Error bars indicate standard deviation in at least two independent measurements. K_D estimated from Rac concentration at 50% saturation; curves are binding isotherms to guide the eye. ΔWRC (containing WAVE1(1-186)) pink triangle, L697D/Y704D_Sra1-miniWRC black square, miniWRC gold diamond, E434K/F626A_Sra1-ΔWRC green circle, R190D_Sra1-ΔWRC blue cross, Δ154_WAVE1-WRC (containing WAVE1(1-154)) cyan inverted triangle. c, WAVE1 meander region. Sra1 residues involved in binding Rac1 and WAVE1 Y151 are gold and blue sticks, respectively. Dashed oval indicates proposed Rac1-binding surface. Phosphorylated WAVE1 residues Tyr 125, Thr 138 and Tyr 151 are red sticks. d, Arp2/3 mediated pyrene-actin assembly assays with miniWRC (green) or miniWRC containing Y151E_WAVE1 (blue) or F686E_Sra1 (red). Control assay (orange) lacked WRC.

Figure 4. Model for cooperative membrane recruitment and activation of the WRC

a, b Electrostatic surface of miniWRC (red to blue = -5 kT e^-1 to +5 kT e^-1), oriented as in Figs. 1a and b, respectively. c, Schematic illustrating proposed WRC orientation at the plasma membrane and cooperative recruitment and activation by Rac and phospholipids. Phosphorylation sites in WAVE1 meander are indicated. It remains unclear what portions of the meander are disrupted by different stimuli.