Structural characterization of intermediates in Sec1/Munc18 protein-catalyzed SNARE assembly

SNARE proteins assemble into membrane-bridging complexes that mediate intracellular membrane fusion. Sec1/Munc18 (SM) proteins serve as essential cofactors by binding cognate R- and Qa-SNAREs in a state of intermediate assembly. Here, we report the crystal structure of a template complex containing the endolysosomal SM protein Vps33 bound simultaneously to its cognate R- and Qa-SNAREs, Nyv1 and Vam3, as well as an improved structure of the R-SNARE-only complex that likely precedes it in the SNARE assembly pathway. In the template complex, short α-helices from each SNARE, together with an α-helical hairpin contributed by the SM protein, form a dynamic SNARE assembly nucleus to facilitate Qb- and Qc-SNARE binding and full SNARE assembly. Notably, Vps33 and the well-studied neuronal SM protein Munc18-1 display similar SNARE assembly nuclei created from different structural elements. The R-SNARE-only structure, along with alanine-scanning mutagenesis, reveals a split mode of Nyv1 binding in which stronger C-terminal interactions provide the majority of the binding energy while weaker N-terminal interactions balance flexibility with SNARE alignment. Taken together, our work defines two early steps in the SM-catalyzed assembly of endolysosomal SNAREs and, more broadly, highlights both conserved and non-conserved features of SM-mediated SNARE assembly pathways.