Crystal Structure of Arp2/3 Complex

Abstract

We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha-helical subunits. We predict that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament.

Nucleotide-Mediated Conformational Changes of Monomeric Actin and Arp3 Studied by Molecular Dynamics Simulations

“…These features also had the highest B-factors or were disordered in crystal structures. 19,21,30 When ATP was added to nucleotide-free Arp3, the cleft did not close, as observed in Arp2/3 complex crystal structures, but opened in a fashion similar to the other Arp3 simulations.…”

“…First, among the nine currently available crystal structures of the Arp2/3 complex, the 5 C-terminal residues of Arp3 that extend beyond the C-terminus of actin show a greater tendency to be ordered when the nucleotide cleft of Arp3 is open than when it is closed. [19][20][21] In the three Arp3 structures with open nucleotide binding clefts, an average of 3.4 of these 5 residues is ordered, while in the three structures with closed clefts, an average of 0.3 of these 5 residues is ordered. This difference suggests that stable interactions of the C-terminal extension with the groove between subdomains 1 and 3 facilitate closure of the barbed-end groove and promote the rigid body motion that opens the nucleotide binding cleft.…”

“…The apo-Arp3, in contrast, is stable and adopts an open conformation in crystals of nucleotide-free Arp2/3 complex. 19,21 These observations, coupled with the fact that the cleft must open to bind or release nucleotide without significant steric clash, led to the proposal that apo-actin opens in the absence of nucleotide. 11 This open state is expected to be on the path to unfolding and destabilization of the protein.…”

“…A crystal structure of the nucleotidefree crystals of Arp2/3 showed Arp3 in an open conformation and Arp2 with subdomains 1 and 2 so flexible that no electron density was present in the maps. 19 Soaking these crystals in ATP caused the nucleotide cleft of Arp3 to close, while soaking them in ADP did not, giving weight to the possibility of an open conformation of ADP-actin. 20 ATP bound subdomains 3 and 4 of Arp2, but the disordered subdomains 1 and 2 did not close around the nucleotide unless the crystal was chemically cross-linked with glutaraldehyde.…”

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

“…Intensive studies had revealed that VCA's ability to bind monomer actin through its V subdomain is critical for actin nucleation (Miki and Takenawa, 1998). The CA subdomain confers to N-WASP its binding ability to the Arp2/3 complex as evidenced by physicochemical assays (Machesky and Insall, 1998;Marchand et al, 2001) and x-ray crystallography and cross-linking experiments (Gournier et al, 2001;Robinson et al, 2001;Zalevsky et al, 2001). Actin polymerization, nucleation, and branching are enhanced in the presence of VCA protein in vitro Machesky et al, 1999;Rohatgi et al, 1999).…”