Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

14-POS Board 14 THE TWO SNARE MOTIFS OF SNAP25 ARE STRUCTURALLY AND FUNCTIONALLY DISTINCT

Katelyn Kraichely 1 ; Connor Sandall 1 ; Volker Kiessling 1 ; Binyong Liang 1 ; Lukas K Tamm 1 ; 1 University of Virginia Health System, Department of Molecular Physiology and Biological Physics, Charlottesville, VA, USA Eukaryotic cells sustain protein and membrane homeostasis through protein-regulated fission and fusion of lipid membranes. Many intracellular membrane fusion events are catalyzed by members of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein family. SNARE proteins form heterotrimers or tetramers that bridge separate membranes and assemble a coiled helical bundle of four 60-70 residue “SNARE motif” domains to induce fusion. The particularly highly regulated fusion events between neuronal synaptic vesicles and the plasma membrane are catalyzed by a SNARE complex composed of syntaxin-1a and SNAP25 (target membrane or “tSNAREs”) on the plasma membrane and synaptobrevin-2 on the vesicle membrane (“vSNARE”). Unlike its synaptic SNARE partners and most other cellular SNAREs, SNAP25 contains two SNARE motif domains connected by a flexible linker and does not contain a transmembrane domain; it is instead associated to the plasma membrane through posttranslational lipidation of its linker domain. These distinctions led us to hypothesize that the unusual arrangement of dual SNARE motifs in SNAP25 may serve a functional role in supporting tightly regulated synaptic exocytosis. We have generated both soluble and membrane associated single SNARE domain constructs of SNAP25 that have allowed us to dissect the structural and functional contributions of each domain. Magnetic resonance experiments revealed distinct structural preferences of the two domains. FRET-based measurements in model membranes showed a correlation between these structural preferences and ability of the individual domains to interact with partner SNAREs. Lastly, a total internal reflection fluorescence microscopy-based fusion assay demonstrated that each domain forms a fusion capable SNARE complex with its cognate SNAREs, though with different efficiency and kinetics. Overall, these data support a model in which the two SNARE domains of SNAP25 serve complementary roles in optimizing fast and regulated membrane fusion at the synapse.

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