(a) Arp3 and Arp2 subunits from snapshots from the last ns of the unphosphorylated (Arp3-gray; Arp2-blue) and phosphorylated T237 Arp2 (tan, red) wild-type simulations are shown following alignment of Cα atoms of subdomains 1 and 2 of Arp3. These and all other structural figures were produced using the molecular graphics program UCSF Chimera [56], except where indicated. The other subunits of the complex from the snapshot of the unphosphorylated simulation, colored as in Fig. 2a, were represented as transparent surfaces in order not to occlude the views of Arp2 and Arp3. (b) RMSD of Arp2 Cα atoms to their initial positions vs. simulation time following alignment of subdomains 1 and 2 of Arp3. (c) RMSD of Arp2 Cα atoms to their positions in the model of the active short-pitch dimer orientation (B. Nolen, personal communication) vs. simulation time following alignment of subdomains 1 and 2 of Arp3. (d) Distribution of root-mean-square deviations (RMSD) of Cα atoms of Arp2 from the MD trajectory to Arp2 atoms in the active short-pitch dimer orientation after alignment of subdomains 1 and 2 of Arp3 over the last 20 ns of simulations. (e) Distribution of number of contacts between Arp3 and Arp2 heavy atoms over the last 20 ns of simulations. In (b)–(e), coloring is as follows: U(black)- unphosphorylated; pT237(blue-gray) – phosphorylated T237 Arp2; pT238(cyan) – phosphorylated T238 Arp2. RMSD of Arp2 Cα atoms in the initial model to their positions in the active orientation is 31.2 Å, and the number of Arp3-Arp2 contacts in the initial model is 35. These values are indicated for reference in the appropriate plots with a green line.

Stereoimages of changes in Arp2-Arp3 orientation in R105A ARPC4 mutant Arp2/3 complex from snapshots from the last ns of the unphosphorylated wild-type(Arp3-gray; Arp2-blue) and the (a) unphosphorylated R105A ARPC4 mutant complex (pink, orange) or (b) phosphorylated T237 Arp2 ARPC4 R105A ARPC4 mutant complex (cyan, magenta) simulations are shown following alignment of Cα atoms of subdomains 1 and 2 of Arp3. The other subunits of the complex from the snapshot of the unphosphorylated simulation, colored as in Fig. 2a, were represented as transparent surfaces in order not to occlude the views of Arp2 and Arp3.

(a) Recombinant Arp2/3 complex WT (rWT) and R105/106A ARPC4 expressed and purified from sf21 cells. (b) Pyrene actin assembly assays comparing nucleation activity of rWT and R105/106A ARPC4 rArp2/3 complex. (c) Pointed end capping assay with actin alone (black), rWT (red) and R105/106A ARPC4 rArp2/3 (green), and R105/106A ARPC4 rArp2/3 pretreated with Antarctic phosphatase (AP-R105/106A ARPC4) (purple).

Stereoimages of the salt-bridge network between Arp2 (pink) and ARPC4 (green) in simulations of wild-type (a) unphosphorylated or (b) phosphorylated Arp2 T237 Arp2/3 complex.

(a) Distribution of root-mean-square deviations (RMSD) of Cα atoms of Arp2 to Arp2 atoms in the active short-pitch dimer orientation (B. Nolen, personal communication) after alignment of subdomains 1 and 2 of Arp3 over the last 20 ns of simulations. (b) Distribution of number of contacts between Arp3 and Arp2 heavy atoms over the last 20 ns of simulations. Coloring is as follows: U(black) – unphosphorylated; R105A(orange) – unphosphorylated R105A ARPC4 mutant; and pT237/R105A(magenta) – Arp2 T237 phosphorylated R105A ARPC4 mutant. Green line represents the corresponding RMSD (31.2 Å) and number of contacts (35) for the unphosphorylated starting model as in Fig. 3.