X-ray scattering study of activated Arp2/3 complex with bound actin-WCA

Abstract

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.

Figure 1. Design and characterization of activated actin-WCA-Arp2/3 complex

(a) From the structure of actin complexes with the W motifs of various cytoskeletal proteins (Chereau et al., 2005), we determined that N-WASP residue Ser 426 could be mutated to Cys and cross-linked to actin Cys 374 with minimal structural disturbance. The actin subdomains are numbered 1 to 4. (b) Non-reducing coomassie-stained gel showing cross-linked actin-WCA, purified Arp2/3 complex, and Arp2/3 complex with bound actin-WCA. (c) Arp2/3 complex with bound actin-WCA (red) has shorter retention time on analytical size exclusion chromatography than Arp2/3 complex alone (blue). Actin-WCA is also shown for reference (black). Retention times are indicated above each peak. (d) The heavier actin-WCA-Arp2/3 complex sediments faster by analytical ultracentrifugation than Arp2/3 complex alone, with sedimentation coefficients of ~11 S and ~8.8 S, respectively. (e) Determination of the molecular masses of Arp2/3 complex with and without bound actin-WCA by FFF-MALS. The measured masses were 224,200 Da for Arp2/3 complex alone (expected value from sequence 223,600 Da) and 273,300 for actin-WCA-Arp2/3 complex (expected 274,060 Da). (f) Time course of barbed end formation of 2 μM Mg-ATP-actin (6% pyrene-labeled) alone (black) or in the presence of 25 nM Arp2/3 complex and 250 nM native WCA (green) or 25 nM actin-WCA-Arp2/3 complex (red). The inset shows the time course of fluorescence increase upon polymerization. Nucleation rates were determined from the polymerization rate, the concentration of actin monomers remaining, and the rate constant for barbed-end elongation (10 μM^-1s^-1).

Figure 2. Structure of inactive Arp2/3 complex

Fitting statistics of the crystal structure of inactive Arp2/3 complex to the scattering data (left). The experimental and computed scattering intensities are displayed as a function of momentum transfer s = 4π sin(θ)/λ, where 2θ is the scattering angle and λ = 1.5 Å is the X-ray wavelength. The scattering data were analyzed in the range 0.01 < s < 0.15 Å^-1, which yields information about the overall shape of the molecule. Higher resolution data were discarded in this analysis due to increased noise. Two orientations of the structure, rotated by 90° and docked into the ab initio SAXS envelope (right). Individual proteins are shown as ribbon diagrams (color scheme: Arp2, cyan; Arp3, green, ARPC1, yellow; ARPC2, purple; ARPC3, maroon; ARPC4, pink; ARPC5, gray).

Figure 3. Scattering statistics for different models of actin-WCA-Arp2/3 complex

(a) Fitting of the model with actin bound at the barbed end of Arp3 to the scattering data (left). In this model Arp2 was moved alone into a filament-like conformation with Arp3 (see also Figure S3f). Two orientations of the model, rotated by 90°, are shown inside the averaged grid envelope resulting from the ab initio SAXS reconstruction (right). A poor fit to the scattering data disqualifies this model. The individual proteins are shown as ribbon diagrams and colored according to other figures of the paper (W motif, red; actin, blue; Arp2, cyan; Arp3, green, ARPC1, yellow; ARPC2, purple; ARPC3, maroon; ARPC4, pink; ARPC5, gray). (b) Fitting of the model with actin bound at the barbed end of Arp2 after activation of Arp2/3 complex by moving Arp2 alone alongside Arp3 (see also Figure S3). The orientation is as in Figure 2. (c) Fitting of the model with actin bound at the barbed end of Arp2 after activation of Arp2/3 complex by moving together Arp2, ARPC1, ARPC4 and ARPC5 (see also Figure S3).

Figure 4. Model of activated Arp2/3 complex

(a) Model of activated Arp2/3 complex with actin bound at the barbed end of Arp2 (as in Figure 3b), showing the most likely path of the WCA polypeptide. The mother and daughter filaments and their respective barbed (or +) ends are tentatively shown for reference (light gray, semi-transparent), but note that the precise organization of the branch is not addressed by this study. Subdomains 1 to 4 of actin and Arp2 and subdomains 2 and 4 of Arp3 are numbered. In the activated structure, Arp2 moves to occupy a filament-like conformation next to Arp3. The first actin subunit, bound to the W motif of N-WASP (red), binds at the barbed end of Arp2. The position of the W motif in the activated complex is derived from the crystal structure of actin-W (Chereau et al., 2005). This location of the first actin and bound W motif imposes constraints on the location of the C and A motifs (magenta and pink). Thus, the hydrophobic cleft between subdomains 1 and 3 of Arp2 is right in the path of the CA polypeptide as it progresses toward Arp2/3 complex. We predict that the helical portion of the C motif (Panchal et al., 2003) binds in this cleft. This idea is also supported by sequence similarity with the W motif and by the fact that tandem W motifs constitute a common architecture among actin filament nucleators (see part b). The C-terminal portion of the C motif runs near the interface between Arp2 and ARPC1, from where the A motif can reach a hydrophobic pocket at the interface between Arp3 and ARPC3, which is consistent with existing binding and cross-linking studies (Kelly et al., 2006; Kreishman-Deitrick et al., 2005; Pan et al., 2004; Weaver et al., 2002; Zalevsky et al., 2001). The conserved Trp 499 of the A motif (yellow) may bind in a hydrophobic pocket at the interface between Arp3 and ARPC3, formed by amino acids Tyr202, Phe 203, Leu 207, Leu 277, Phe 287 of Arp3 and Phe 54 and Phe 163 of ARPC3. (b) Alignment of the WC motifs of N-WASP with tandem W motifs of proteins involved in actin assembly. Conserved amino acids are colored according to their chemical characteristics (green, hydrophobic; blue, basic; magenta, prolines; brown, glycines; yellow, small conserved amino acid). The WC motifs of N-WASP align particularly well with the G- and F-actin binding (GAB and FAB) motifs of vasodilator-stimulated phosphoprotein (VASP) (Ferron et al., 2007). Since FAB binds F-actin, it appears likely that the C motif binds Arp2 within Arp2/3 complex (see text). Tandem W motifs are emerging as a common architecture in actin filament nucleation. Spire (Quinlan et al., 2005), Cobl (Ahuja et al., 2007) and the Vibrio proteins VopL (Liverman et al., 2007) and VopF (Tam et al., 2007) are recently discovered filament nucleators that utilize tandem W motifs to assemble actin monomers into polymerization seeds. Of note, Cobl, VopL and VopF contain only three W motifs and present a long Pro-rich linker between the second and third W motifs. Although this linker in spire presents all the signature features of a W motif, it is also rich in Pro residues. By analogy with these proteins, we predict that WC constitutes a specialized form of tandem W motifs, where C has evolved specificity for Arp2, allowing for the combination of the nucleation and branching functions by Arp2/3 complex. Sequence accession numbers are: Cobl (Mouse, CAI36023); Spire (Drosophila melanogaster, Q9U4F1); VopF (Vibrio cholerae, AAZ32252); VopL (Vibrio parahaemolyticus, NP_800881); VASP (Human, P50552); N-WASP (Mouse, CAC69994).