Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors

Abstract

Arp2/3 complex initiates the growth of branched actin‐filament networks by inducing actin polymerization from the sides of pre‐existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three‐dimensional structures at ∼2 nm resolution of Arp2/3 complex with three different bound NPFs: N‐WASp, Scar‐VCA and cortactin. All of these structures adopt a conformation with the two actin‐related proteins in an actin‐filament‐like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin‐filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.

Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

“…One possibility is that the cortactin NtA harbors sequences unique from the WASP C region that stimulate conformational changes in the complex. In support of this, single particle reconstructions of electron microscopy images of cortactin-bound Arp2/3 complex show Arp2 and Arp3 in a filament-like conformation distinct from the splayed conformation observed in crystal structures of inactive Arp2/3 complex (63). FRET experiments by Goley et al (64) showed that unlike CA, an NtA construct containing residues 1-39 does not stimulate conformation changes in the complex, indicating that NtA residues important for these changes may be C-terminal to residue 39.…”

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

“…The formation of the initial closed Arp2/3-filament complex (A 2 F) is reversible, but the Arp-filament interaction is stabilized by subsequent activation processes (13,17), which may include binding of actin monomers (13,15), changes in the nucleotide state of Arp2 and/or Arp3 (16,25), additional Arp2/3 complex conformational changes (6,10,17), or alteration of the mother filament structure or curvature (6,28,31). Because the experiments were conducted at either zero or maximally activating concentrations A B actin-AF488 Arp2/3 binding heat map merge of VCA, the scheme does not explicitly show VCA binding steps or define the occupancies of the multiple VCA-binding sites (10,14,15). Instead, we make the simplifying assumption that the arrangement of states is the same in both cases and, consequently, the effect of VCA can be incorporated by changing the apparent rate or equilibrium constants.…”

“…Nucleation of actin filament branches involves large-scale conformational changes in the Arp2/3 complex (6-10), binding of one or more verprolin homology, central, and acidic (VCA) domains (10)(11)(12)(13)(14)(15), recruitment of one or more actin monomers (2,16), and formation of a stable association of the complex with a mother filament (6). The ordering, dynamics, and regulation of these individual reaction steps in the overall process of branch nucleation have been subjects of intense interest (13,15,17,18).…”

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

“…How WASP proteins activate the Arp2/3 complex is unknown, but available data suggest they play multiple roles in the branching nucleation reaction. For instance, several lines of evidence indicate that the CA segment of WASP proteins stimulates a conformational change in the complex important for activation (11)(12)(13)(14)(15). In addition, WASP-VCA recruits actin monomers to the complex through its V region.…”