Conformational Changes of Arp2/3 Complex in the Branched Actin Nucleation Pathway

Abstract

Branched actin networks play an important role in cellular processes ranging from cell motility, endocytosis, and adhesion. The Actin-related protein 2/3 (Arp2/3) complex nucleates actin branches from the sides of existing actin filaments. Arp2/3 complex is highly regulated and requires association with ATP, actin monomers, actin filaments and a class of proteins called nucleation promoting factors (NPFs) to undergo an activating conformational change where the actin-related subunits, Arp2 and Arp3, arrange into a filament-like conformation that templates a new actin branch. While some progress has been made, the individual roles of each of these factors on the activating conformational change is poorly understood. In addition, it is still unclear how Arp2/3 complex is held in its inactive state, which is vital for understanding how activation occurs. In this dissertation, we dissect key interfaces in Arp2/3 complex that are responsible for holding it in an inactive state, and specifically evaluate the roles of ATP and WASP, the canonical NPF, in the activating conformational change of Arp2/3 complex.

In chapter II, we investigated the contacts made between the Arp2 and Arp3 subunits in their inactive state, and the role of ATP in stimulating the active conformation. We found that two key interfaces, the αE/αF loop in Arp2 and the C-terminus of Arp3, a conserved extension not present in actin, are vital for holding Arp2/3 complex in its autoinhibited state. Evaluation of the role of ATP demonstrated that binding of ATP is required for the activating conformational change and displaces the Arp3 C-terminus, an important step in destabilization of the inactive state.

In chapter III, we investigated the mechanism of WASP-induced conformational changes using an engineered crosslinking assay that only forms crosslinks when Arp2/3 is in its active conformation. We discovered that many WASP-related proteins are capable of stimulating this conformational change through a mechanism that involves displacement of the Arp3 C-terminus. Interestingly, purified Arp2/3 complex crosslinked in the active conformation was hyperactive compared to WASP-mediated activation, demonstrating that WASP activation limits nucleation and that actin monomer delivery is not required for nucleation.

This dissertation contains unpublished co-authored material.