An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability

Abstract

The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.

Fig. 1.

Protein-protein docking simulations identify a putative mother filament binding site on the Arp2/3 complex. (A) Surface rendering of the structures of an ARPC2/ARPC4 heterodimer and a portion of the actin filament, with residues predicted to form salt bridges highlighted in red (acidic) and blue (basic), and the surface predicted to be within 4 Å of the other binding partner in yellow, based on molecular dynamics and protein-protein docking simulations. Each structure is rotated 90° away from the other to show the binding interfaces. (B) Structures of the whole Arp2/3 complex with the predicted mother filament binding interface highlighted in yellow. (C) Structure of the inactive Arp2/3 complex (12) docked onto a mother filament in the orientation dictated by the position of ARPC2/ARPC4 in the top-scoring model from docking simulations.

Fig. 2.

Residues on ARPC2 and ARPC4 identified by molecular docking and homology modeling lie on a surface that is critical for Arp2/3 complex activity. (A) Surface rendering showing the location of amino acid residues selected for mutagenesis (opaque and color coded) based on molecular docking (blue), homology modeling (green), or both (magenta). (B) Surface rendering showing the correlation between the location of mutations on the surface of ARPC2/ARPC4 and the severity of the actin nucleation defects. The surface that is predicted to be within 4 Å of actin by protein-protein docking is opaque and yellow, and the side chains of mutated amino acids are shown in space filling representation and are color coded as follows: orange, severely defective; purple, moderately defective; green, unaffected.

Fig. 3.

Mutations in charged surface residues on ARPC2 and ARPC4 cause a range of actin nucleation defects. (A) Pyrene-actin polymerization assays with wild-type (WT) and mutant Arp2/3 complexes. Reactions contain 3 µM actin, either alone (actin) or with 20 nM WT or the indicated mutant complexes (see Table S2 for abbreviations) and 200 nM GST-WASP-WCA. Mutant complexes with similar activity levels are grouped together and colored according to the scheme in Fig. 2B. (B) Concentration of barbed ends generated by WT and mutant Arp2/3 complexes as a function of Arp2/3 concentration.

Fig. 4.

Mutant Arp2/3 complexes undergo expected conformational changes on activator binding, but fail to bind F actin with high affinity or form long-lasting branches. (A) Box and whiskers plot of normalized FRET/CFP ratio of WT, 4DKK, and 4RED Arp2/3-FRET complexes in the presence of Mg²⁺, Mg-ATP, or Mg-ATP and GST-WASP-CA. The middle line of each box indicates the median, and the top and bottom lines represent the third and first quartiles (n = 6). Whiskers indicate maximum and minimum measurements. (B) Percentage of WT (black) or 4DKK (orange) Arp2/3 complex found in the pellet fraction after high-speed centrifugation over a range of actin concentrations. Dissociation constants (K_ds) calculated from the resulting curves are indicated. Data are the mean ± SEM (n = 6). (C) Images of branches formed by WT and the 4DKK complexes. Scale bar 2 µm. (D) Graph of the fraction of the initial branching frequency (normalized such that the branch frequency at t = 0 is 1) vs. time after initiating nucleation/branching for WT (black) and 4DKK (orange). Data are the mean ± SEM (n = 3).