Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

55-POS Board 19 CONNECTING STRUCTURE AND FUNCTION: MOLECULAR CHARACTERIZATION OF THE ESSENTIAL PRESYNAPTIC PROTEIN UNC-13 USING GENETIC AND BIOCHEMICAL APPROACHES Ziasmin Shahanoor 1 ; Kirill Grushin 2 ; Murugesh Padmanarayana 1 ; Noa Lipstein 3 ; James E Rothman 2 ; Jeremy S Dittman 1 ; 1 Weill Cornell Medicine, Biochemistry, New York, NY, USA 2 Yale School of Medicine, Cell Biology, New Haven, CT, USA 3 Liebniz Institute for Molecular Pharmacology, Berlin, Germany Chemical synaptic transmission, vital for proper nervous system function, requires a precise sequence of molecular steps that culminate in the formation of SNARE complexes. Recent studies have placed the essential multidomain protein UNC-13 (Munc13-1 in rodents, UNC13A in humans) at the hub of the synaptic fusion apparatus, regulating a host of functions including release site-building, synaptic vesicle (SV) positioning, and SNARE assembly. However, a comprehensive mechanistic understanding for these UNC-13 functions remains elusive. Based on data from our lab and others, we propose that sequential functions of UNC-13 moving from SV capture to tight docking, priming/SNARE assembly, and finally to post-priming enhancement of release probability map onto the UNC-13 protein structure from its C-terminus towards its centrally located C1 domain. Using C. elegans as a model nervous system, we recently characterized a novel domain at the C-terminus of UNC-13 (HC2M) required for SV docking and are currently testing the hypothesis that HC2M binds SVs via a protein-protein interaction. The neighboring C-terminal half of the MUN domain has been proposed to mediate intra- and intermolecular interactions with neighboring UNC-13 proteins to form oligomeric structures that function in concert to coordinate SV capture and SNARE assembly during the docking and priming process. Mutations that disrupt these interactions severely impair neurotransmitter secretion in the worm. Finally, we previously suggested that the C1-C2B tandem domain neighboring the UNC-13 MUN domain serves as an autoinhibitory regulator of SV fusion. We observed that a dominant human mutation located within a crucial hinge between C1-C2B and the MUN domain is associated with a gain-of-function synaptic phenotype in heterozygous worms, supporting the notion that small assemblies of UNC-13 monomers act in concert to promote and inhibit SV fusion. Connecting the functional and structural aspects of UNC-13 will sharpen our understanding of the molecular choreography underpinning neurotransmitter release.

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