Investigating the emergent dynamics of actin filament branch formation and aging using a multiscale computational approach

PROJECT SUMMARY Actin-binding proteins (ABPs) direct and meticulously regulate several actin-related cellular processes. Actin- related protein (Arp)2/3 complex is an ABP that nucleates “daughter” branches from existing “mother” filaments which push against the plasma membrane to facilitate endocytosis and motility. Dysfunction is implicated in cell invasion and metastasis in which Arp2/3 complex and branch stabilizers such as cortactin are overexpressed. In filamentous (F-)actin, the bound nucleotide state and rates of transition between states function as a biological clock to mark the age of the filament. Like F-actin, the Arp2 and Arp3 subunits of Arp2/3 complex bind ATP which is necessary for the nucleation of the daughter filament. However, the role of ATP hydrolysis and subsequent phosphate release in both Arp subunits on the aging and dissociation of actin filament branches is still being explored. In this work, I propose using novel computer simulations to obtain a mechanistic understanding of three key processes in the aging of actin branch filaments, focusing on the role of the nucleotide state bound to Arp2 and Arp3 on branch aging and dissociation. ATP hydrolysis is important for branch dissociation, but not necessary for branch formation. Differences in the propensities for ATP hydrolysis in Arp2 and Arp3 based on the protein source, the presence of the daughter and mother filaments, and Arp2/3 complex activation method indicate that multiple factors may influence hydrolysis rates. Importantly, the outlined factors consist of large protein complexes which preclude conventional quantum mechanics/molecular mechanics (QM/MM) methods. I will develop a multiresolution computational framework to obtain thermodynamics, kinetic, and mechanistic insights into ATP hydrolysis in the Arp2 and Arp3 subunits, respectively, in the context of the coarse-grained protein environment. I will focus on the role of the mother and daughter filament in facilitating the rearrangement of key amino acids in the active sites of Arp2 and Arp3. Phosphate release in Arp3 substantially decreases the mechanical stability of branches under force. The release process itself is very slow with lifetime estimates ranging from <1 minute to >80 minutes depending on the source. I will employ novel enhanced sampling methods with all-atom molecular dynamics (MD) simulations to investigate the kinetics and mechanism of release in Arp2 and Arp3. Differences in amino acids of the exit channel and backdoor gate of F-actin, Arp2, and Arp3 may influence the relative rates of this process. Finally, debranching under force will be modeled with MD simulations to obtain a molecular-level understanding of the mechanism and its dependence on the nucleotide state of Arp2 and Arp3. This is important to understand because it has direct implications for the fate of the branch and its ability to regenerate. Cortactin will be incorporated into the model to determine how branch stabilizers may influence the debranching mechanism. The outcome of this work will provide a cohesive mechanistic understanding of ATP hydrolysis, phosphate release, and branch dissociation which is critical to evaluate the role of Arp2 and Arp3 in growth and turnover of branches.