Structural and biochemical characterization of two binding sites for nucleation-promoting factor WASp-VCA on Arp2/3 complex

Abstract

Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites ( K d s of 0.13 and 1.6 μM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that ( i ) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, ( ii ) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and ( iii ) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.

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

“…These techniques allowed us to directly measure key features of Arp2/3 complexmediated branch formation, including Arp2/3 binding and dissociation rates, branch formation efficiency, and activation time, and to determine how these parameters are affected by VCA. Earlier studies observed actin only (4, 5), or used indirect bulk methods based on fluorescence enhancement of pyrene-actin and/or pyrene-Arp2/3 complex (15,17,18). Our activation timedelay results are roughly consistent (within ∼10-fold; SI Materials and Methods) with those derived from kinetic modeling based on earlier observations.…”

“…Our activation timedelay results are roughly consistent (within ∼10-fold; SI Materials and Methods) with those derived from kinetic modeling based on earlier observations. However, our observations reveal that binding and most dissociation of Arp2/3 complex from filaments is ∼100 and ∼1,000-fold faster than inferred previously (15). Conversely, branch formation is extremely inefficient (0.5-1.5%), in contrast to the ∼100% efficiency that would be implied if dissociation were slow.…”

“…The vast majority (∼98%) of Arp2/3-filament complexes were unsuccessful in nucleation and dissociated from the filament in two shorter lifetime components, with time constants of τ 1 = 2 s (∼90% of events) and τ 2 = 10-15 s (∼8% of events). These observations suggest that most Arp2/3-filament complexes are >10 3 -fold more kinetically unstable than was inferred from previous experiments (15,18), and that the simple Only ∼2% of the Arp2/3-filament complexes were in the longest-lived component of the lifetime distribution. The τ 3 time constants (Fig.…”

“…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). Elucidating the mechanism of this elaborate process has been challenging using conventional bulk biochemical experiments, because they average together the signals from multiple reaction steps.…”