Investigating the Interaction Between the Sec1/Munc18 (SM) Protein Vps33 and its Qa- SNARE Vam3 and R-SNARE Nyv1

Abstract

SNAREs zipper together to form four-helix membrane-bridging complexes that promote membrane fusion. SNARE complex formation is regulated by Sec1/Munc18 (SM) proteins. SM proteins interact with SNAREs, but exactly how they regulate SNARE complex assembly is not well understood. Previously, our lab reported separate crystal structures of the SM protein Vps33 bound to its R-SNARE Nyv1 and to its Qa-SNARE Vam3. Superposition of the two structures suggested that Vps33, and potentially other SM proteins, could serve as templates to bring the R- and Qa-SNAREs together to form a half-zippered SNARE complex. However, no structure of an SM protein bound simultaneously to its Qa- and R-SNAREs has been reported for any SM protein. A structure of the ternary complex will reveal whether and, if so, how Nyv1 (R-SNARE) and Vam3 (Qa-SNARE) influence each other’s binding to Vps33 as they initiate SNARE complex assembly. In this thesis, we explored a novel strategy of purifying Nyv1 and Vam3 as disulfide-linked heterodimers to obtain a crystal structure of the ternary complex. This strategy has been previously shown, through single-molecule force spectroscopy, to be compatible with template complex formation. Using this approach, we identified three conditions that showed crystal growth. Diffraction data were collected from optimized crystals of one of the conditions. Biochemical analyses were performed to better characterize the interaction between Vps33 and its SNAREs. Addition of Vps33 enhanced the formation of covalent Nyv1/Vam3 heterodimers. This provides new evidence in support of the model that Vps33 serves as a template for the assembly of Nyv1 and Vam3. In addition, isothermal titration calorimetry assays were performed to estimate the dissociation constants between Vps33 and Nyv1 and Vps33 and Vam3. Taken together, our findings suggest that engineered disulfide bonds may represent a promising strategy for structurally characterizing SNARE assembly pathways.