Project summary/abstract: The goal of this proposal is to elucidate novel mechanisms of Arp2/3 complex inhibition by two protein inhibitors, Arpin and Coronin, as dysregulation of this system cause muscle diseases such as cardiomyopathyand underpin the invasive nature of cancer metastasis. Although mechanisms of inhibition have been widely studied, inhibition has remained largely unexplored due to little progress towards the classification of direct inhibitors of Arp2/3 complex. In a major breakthrough, I biochemically and structurally characterized Arpin as an inhibitor of Arp2/3 complex by providing the first structure of Arpin bound to the complex. This seminal finding provided the necessary insights to guide biochemical experiments towards precisely describing the mechanism of inhibition. I found that Arpin binds the complex in a similar manner to activating proteins NPFs via structurally homologous C-termini, with distinct but important differences that ultimately classify each protein as an inhibitor or activator, respectively. In aim 1, I will continue this line of investigation by exploring the mechanism of inhibition through a crosslinking assay that monitors conformational changes of Arp2/3 complex that will lead to the discovery of a novel mode of inhibition. I will also investigate how post-translational modifications on Arpin influence Arp2/3 complex binding and inhibition, also studying in parallel the uncharacterized globular domain of Arpin through structural and cell-based studies (accomplished through collaboration). In aim 2, I will apply the skills and knowledge learned from aim 1 to investigate the long-standing proposed inhibitor Coronin, as there exists conflicting reports on their activity towards Arp2/3 complex. I will take a representative member of the canonical mammalian Coronins (Coronin 1A) and test its ability to regulate Arp2/3 complex function using a wide array of biochemical, biophysical, and structural approaches. In aim 3, I will focus on the remaining and most unique member of mammalian Coron ins, Coronin 7. Coronin 7 contains a potential Arp2/3 complex binding domain at the C-terminus similar to NPFs and Arpin, but divergent from canonical Coronins containing a coiled-coil. If this region interacts with Arp2/3 complex or branched actin networks is not known and is a main focus in this proposal. Altogether, this work addresses gaps of knowledge in regard to inhibition of Arp2/3 complex, a process that comprises half of the overall regulatory system that governs cell motility and vital intracellular processes. The success of my aims depends mainly on my ability to executive the proposed experiments and produce pure Coronin proteins, which I demonstrate in preliminary studies. My studies on Arpin position me to ask unique questions on Arp2/3 complex inhibition investigated in this proposal. Accomplishment of the aims should have a far-reaching impact, providing a shift in perspective on inhibitory mechanisms of...