UC San Diego

Synaptic function and integrity in the nervous system requires the expression of synaptic adhesion molecules (SAMs) linking pre- and post-synaptic sites. Studies indicate that SAMs participate in the formation, maturation, function and plasticity of synaptic connections, and thus are essential for trans-cellular signaling. Alterations in SAMs lead to susceptibility to neurological diseases including the autism spectrum disorders, schizophrenia, and addiction. Neurexins compose a family of highly polymorphic type I transmembrane proteins that are expressed on the pre-synaptic membrane at excitatory glutamatergic and inhibitory GABAergic synapses. Beta-neurexins have a single folding domain in the extracellular region, while alpha-neurexins have a larger extracellular region containing nine independently folding domains and multiple protein interaction sites. They both function as adhesion molecules through a trans- synaptic complex with post-synaptic neuroligins. Synaptogenesis and synapse function requires the precise assembly of pre- and post-synaptic protein complexes. The work described herein uses structural and biophysical techniques to discern the molecular properties of the neurexin and neuroligin proteins that mediate their complex formation. The first aim included solving the X- ray crystal structure of the beta-neurexin:neuroligin complex, which showed a stable neuroligin dimer and two monomeric beta-neurexin molecules bound on either side of the dimer. This structure revealed the molecular adhesion properties of the complex, including a Ca²⁺-coordination site. Alpha- and beta-neurexins have the same binding domain for neuroligins, yet they are likely to act as functionally distinct molecules in the synapse. To consider their distinctive adhesion properties, as well as how the multi-domain alpha-neurexins assemble in the limited space of a synapse, the second and third aims were directed at solving the 3D structure of alpha-neurexin. First, small angle X-ray scattering and single-particle negative-stain electron microscopy provided information on the overall domain organization and flexibility of the protein. This work led to the high-resolution crystal structure of a major portion of the alpha-neurexin extracellular region. The crystal structure reveals molecular details that suggest a multi-functional mechanism of the alpha-neurexin extracellular region in the synapse. Overall, this work contributes to the understanding of how synapses assemble through a complex network of protein-protein interactions and provides structural templates for the development of molecular tools to study function and potentially therapeutic applications