Project Summary Excitatory synapses exhibit characteristic proteinaceous microcompartments at the membrane known as the post-synaptic density (PSD). The PSD is richly organized into multi-protein complexes composed of glutamate receptors, scaffolds, cytoskeleton, and signaling effectors. The dynamic reorganizations of the higher-order architecture and composition of PSD signaling complexes underlies synaptic plasticity, learning, and memory. Dissecting the interactions and allosteric communication mechanisms of the PSD is a prerequisite for understanding the molecular underpinnings of synaptic plasticity. Synaptic signaling complexes are large and highly dynamic, problematic targets for mainstay structural biology approaches. The overarching goals of this project are to surmount the challenges of structural characterization of PSD signaling complexes by applying a battery of protein footprinting, spectroscopies, chemical tools, and proteomics approaches to probe protein structure in biologically relevant milieu. This project focuses on the architecture, activation mechanisms, and scaffolding roles of SynGAP, an abundant PSD GTPase-activating protein. SynGAP is critical to brain development, long-term potentiation, and spatial learning. Importantly, SynGAP mutations in humans are associated with autism spectrum disorders, schizophrenia, and intellectual disability. Despite its ubiquity and central signaling role, the architecture and signaling mechanisms of SynGAP remain largely unknown. Defining the molecular mechanisms of SynGAP signaling is critical for understanding the basis of brain disorders caused by SynGAP dysfunction. Hybrid structural biology approaches will be integrated to build a structural model of the multi-domain holoenzyme. These approaches are uniquely amenable to characterizing dynamic protein interfaces and conformational changes in solution. Phosphorylation-induced conformational changes will be defined to determine activation mechanisms. SynGAP apparently regulates opposing pathways dictating synaptic strength via dual specificity toward both Ras and Rap. Specificity switching mechanisms will be revealed by mapping structural determinants of Ras and Rap interactions. By dissecting the structural basis for SynGAP function the proposed research will be a vital contribution to the ongoing movement to characterize the molecular architecture of the PSD. Resolving SynGAP signaling complex structure and mechanisms will clarify the basis of SynGAP-linked neuronal disorders and spur the development of future therapeutics.