Arp2/3 complex is bound and activated by two WASP proteins

Abstract

Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott-Aldrich syndrome protein (WASP) family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.

Fig. 1.

Two VCAs bind Arp2/3 complex, at distinct sites. Sedimentation velocity analytical ultracentrifugation experiments of 0.54 μM Arp2/3 complex in 9.9 μM VCA* (see Fig. S1A in SI Appendix) were collected using absorbance at 496 nm and interference signals. See Fig. S1 B and C in SI Appendix for sedimentation data and fits. (A) The two signals were used to determine c_k(s) distributions for the VCA* and Arp2/3 complex. The shaded regions are integrated to find the quantity of Arp2/3 and VCA* that cosediment (indicated in panel). See Table S1 in SI Appendix for a summary of observed stoichiometries. (B) The observed ratio of bound VCA* to Arp2/3 complex when the indicated concentrations of cortactin NtA domain were added. A and B are adapted with permission from ref. .

Fig. 2.

Two actins are delivered by two VCAs to Arp2/3 complex. Sedimentation velocity analytical ultracentrifugation experiments with Arp2/3 complex, N-WASP VCA (second V-region only), and actin. (A and B) c(s) distributions were derived for the indicated combinations of materials. Data were collected at 50,000 rpm. Actin stoichiometry estimates made use of integrated signal intensities for the peaks at 3.8 S in “VCA + Actin” and “Arp2/3 + Actin + VCA,” at 9.2 S in “Arp2/3,” and 11.4 S in “Arp2/3 + Actin + VCA.” “Arp2/3” and “Arp2/3 alone” in A and B are the same data. (C) Calculations for the determination of stoichiometry. Signals are derived from integration of data shown in A and B. See also Fig. S2 in SI Appendix for determination of stoichiometry through estimation of the VCA-actin-Arp2/3 assembly molecular weight.

Fig. 3.

Label transfer from CA to Arp2/3 complex. (A) Arp2/3, E491C and E498C N-WASP CA label transfer donors were mixed as shown and illuminated. Where indicated, cortactin NtA domain was added as a competitor. Biotinylated Arp2/3 subunits were visualized using Neutravidin-HRP and identified by comparison to the Memcode Blue stained blot (lane marked MB). Nonspecific cross-reaction with ArpC3 is indicated with “ns.” Label transfer to ArpC3 results in a mobility shift, see Fig. S3 D and E in SI Appendix. (B) Label transfer from N-WASP CA mutants (listed beneath the CA sequence) was scored qualitative for biotinylated band intensity (see Fig. S3B in SI Appendix). Cortactin NtA dependent changes are indicated at right. No labeling of ArpC2, ArpC4, or ArpC5 was observed. (C) Label transfer experiments using Cys-NtA and NtA-Cys as donors. “MB” and “ns” as described in A. (D) N-WASP CA, where W503 was substituted with BPa, was mixed with Arp2/3 complex, and illuminated when indicated. Coomassie brilliant blue staining of products, new bands are indicated by an asterisk. (E) Cross-linking products from D are identified by blotting. Lane 1, molecular weight markers. Lane 2, 4, and 6 are a mixture of CA and Arp2/3 following illumination. Lanes 3 and 5 are Arp2/3 alone. Lane 2 was probed for biotinylated proteins (as in A). Lanes 3 and 4 were probed for Arp3 (using an anti-Arp3 antibody). Lanes 5 and 6 were probed for ArpC1 (using an anti ArpC1B antibody).

Fig. 4.

Activation of Arp2/3 complex by deletions of VCA–VCA. (A) Cartoon representation of the VCA–VCA materials. VCA is from human WASP. (B) Actin polymerization rates found in Arp2/3 dependent actin polymerization assays with the indicated concentrations of the VCA materials described in A. (C) Actin polymerization rates found in Arp2/3 dependent actin polymerization assays with VCA dimers with different length N-terminal linkers. Horizontal dashed line is the activity of unstimulated Arp2/3. Activity assays were performed with 10 nM Arp2/3, 4 μM Actin (5% pyrene labeled) in KMEI150, and are the average of three to six repeats. Error bars are the 1σ standard error on the mean, and in many cases are smaller than the symbol. See also Fig. S4 in SI Appendix for VCA dimer sequence details, measurements of the affinity of the materials in A for Arp2/3 and for titrations of the N-terminal linker variants.

Fig. 5.

Asymmetric engagement of Arp2/3 complex by VCAggsVCA. (A) Cartoon representation of the VCAggsVCA constructs produced. VCAggsVCA materials were produced with H472C (native WASP sequence numbers) in either the N-terminal (top line) or C-terminal (second line) VCA. Deletion of the N-terminal or C-terminal V-region results in CAggsVCA and VCAggsxCA materials, respectively. (B) Label transfer experiments from the H472C mutant VCAggsVCA materials to Arp2/3 in the absence or presence of actin. (C) Actin polymerization rates in Arp2/3 dependent actin polymerization assays stimulated with the indicated VCA materials, 10 nM Arp2/3, and 4 μM actin (5% pyrene labeled) in KMEI150, and are the average of three to six repeats. Error bars are the 1σ standard error on the mean, and in many cases are smaller than the symbol. See also Fig. S5 in SI Appendix for the activity of VCAggsVxA and VxAggsVCA and the sequence of VCAggsVCA.

Fig. 6.

Proposed binding sites for VCA on Arp2/3 complex. Structural models of the VCA-binding sites on Arp2/3. Models are constructed as described in the text using the information summarized in Table S2 in SI Appendix. (A–C) Cartoon depiction of CA binding orientation shown in thick black lines, on top of the inhibited Arp2/3 complex shown in surface representation. Subunits are colored as: Arp3-orange, Arp2-red, ArpC1-green, ArpC2-cyan, ArpC3-magenta, ArpC4-blue, and ArpC5-yellow. Conserved patches (28) are indicated by color changes to the surface. C-2 (on Arp2) and A-2 (on Arp3) are indicated by black surface. Conserved basic residues of M-3 (on ArpC1) and A-1 (on Arp3) are shown in blue, highly conserved, nonbasic residues of M-3 are shown in white surface. (A) The proposed Arp3 binding site. (B) A reference orientation of Arp2/3, with labeled subunits. (C) The proposed Arp2/ArpC1 binding site. (D) Delivery of actin to Arp2/3 in a long-pitch dimer orientation relative to Arp3 results in a large steric clash with Arp2. (E) An Arp2/3 complex model with Arp2 moved to a short-pitch actin dimer position with respect to Arp3. (F) A model for Arp2/3 bound to two VCA peptides and two actins. Actin monomers have been added in a long-pitch positions relative to Arp2 and Arp3. Arrows indicate the rotation of molecules relative to the reference view of the Arp2/3 complex in panel B. Panels E and F are in the same orientation.

Fig. P1.

The nucleation of new actin filaments is a tightly regulated process. The actin nucleation factor, Arp2/3 complex is a seven-membered protein machine (shown as a multicolored assembly, with subunit names indicated in black lettering). Arp2/3 complex is activated by VCA peptides (indicated in black) of the WASP family of proteins. Described in this study is a structural model of the nucleation process, in which two of these peptides deliver two actin monomers (in gray) to Arp2/3 complex.