The long-term goal of this project is to develop an in-depth understanding of the structure of CaM-kinase II and to relate that structure to the kinase's diverse neuronal functions. Two main goals motivate the present proposal. The first goal is to resolve the structure of CaM-kinase II at a resolution that will enable visualization of the molecule as it transitions through different states of activation. We aim to define the underlying mechanisms and structural consequences of Ca2+ /CaM-binding and autophosphorylatlon that confer unique and essential properties to this multifunctional kinase (e.g., Ca2+/CaM-lndependent activity, CaM trapping, Ca 2+ frequency encoding). The second goal is to identify binding sites, and the global structural consequence of interactions between CaM-kinase II and substrates as well as other proteins that bind distal to the active site. This goal is driven by a hypothesis that the oligomeric structure of CaM-kinase II is utilized as a scaffold at synapses on which other proteins can be assembled. The proteins selected for this study are localized to synapses and interact with the kinase in potentially unique mechanisms. Overall, five specific aims will be pursued. Aim 1 will be the finalization of low-resolution structures of Ca2+/CaM-bound CaM kinase II and the autophosphorylated form of the kinase. Aim 2 will be to apply state-of-the-art methodologies to attain high-resolution (<10 Angstroms) structures of the native, Ca2+/CaM-bound, and autophosphorylated forms of CaM-kinase II. Finally, we will determine the structures of CaM-kinase II in association with synapsin 1 (Aim 3), the NR2B subunit of the NMDA receptor (Aim 4), and densin-180 and alpha-actinin (Aim5).