Mechanism of Activation of the Arp2/3 Complex by Nucleation Promoting Factors

Abstract

Arp2/3 complex is a ubiquitous actin filament nucleator comprised of seven proteins, two of which (Arp2 and Arp3) are related to actin. By itself Arp2/3 complex is inactive. It is activated by nucleation-promoting factors (NPFs) that contain a C-terminal WCA sequence. By bringing together actin and Arp2/3 complex NPFs catalyze a reaction that leads to the formation of a new actin filament branch bound at 70° angle to the side of a pre-existing filament. It is generally believed that the activation mechanism involves a conformational change within Arp2/3 complex that brings the two Arps close to one another, analogous to two parallel actin subunits of the actin filament. The two Arps, together with the actin monomer(s) delivered by NPFs, form a polymerization ‘seed’. However, the exact mechanism of activation remains unknown, and there is disagreement as to which subunits of Arp2/3 complex interacts with NPFs and how. Currently, there are two competing models of activation. The first model is based on small angle X-ray scattering of Arp2/3 complex bound to WCA region of the NPF and actin. According to this model only one NPF binds Arp2/3 complex and delivers the first actin monomer of the branch at the barbed end of the Arp2. The second model assumes that two NPF molecules are required for optimal activation, with the first actin monomer being delivered at the barbed end of Arp3. The studies in support of the latter mechanism do not directly analyzed binding of NPF carrying actin to Arp2/3 complex. Here, we present a study of the polymerization mechanism of the Arp2/3 complex that takes into consideration this critically missing factor, by using ITC and fluorescence to investigate the interaction of NPFs carrying actin with Arp2/3 complex.