Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor

Area of Science:

• Cell Biology
• Biochemistry
• Structural Biology

Background:

• The Arp2/3 complex is essential for generating branched actin networks, driving critical cellular processes like motility and cytokinesis.
• It consists of seven proteins, including Arp2 and Arp3, and interacts with existing actin filaments at branch junctions.
• Arp2/3 complex exists in inactive (end-to-end Arp interaction) and active (side-by-side Arp interaction) conformations, with cofactors like NPFs and ATP influencing this transition.

Purpose of the Study:

• To elucidate the precise contribution of various cofactors in the activation of the Arp2/3 complex.
• To determine the structural basis of Arp2/3 complex activation by visualizing a transition state.
• To understand the coordinated role of activating cofactors in modulating Arp2/3 complex conformation and function.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structure of the Arp2/3 complex in a transition state of activation.
• A constitutively dimeric NPF, N-WASP, was engineered and fused to CapZ to stabilize the complex.
• Structural analysis focused on the conformations of Arp2, Arp3, and actin within the transition state.

Main Results:

• A 3.32-Å resolution cryo-EM structure of the Arp2/3 complex in an intermediate activation state bound to a dimeric NPF was obtained.
• The structure revealed intermediate conformations of Arp2, Arp3, and actin, distinct from both monomeric and filamentous states, with only actin hydrolyzing ATP.
• The transition complex exhibited a kinetic shift towards the active short-pitch conformation and increased affinity for mother filaments compared to the inactive complex.

Conclusions:

• The study provides a structural snapshot of Arp2/3 complex activation, revealing intermediate conformations of its core components.
• It demonstrates how NPFs, actin monomers, and mother filament binding collectively contribute to Arp2/3 complex activation.
• These findings clarify the coordinated mechanism by which diverse cofactors drive the transition to the active, branched actin network-forming state.