Investigating SM-SNARE Protein Interactions Within the Vps33-Vam3-Nyv1 Complex

Abstract

Eukaryotic cells use vesicles to transport cargo among different cellular compartments via membrane fusion. Membrane fusion is stimulated by the assembly of cognate Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor (SNARE) proteins into a four-helix membrane-bridging complex. This assembly is modulated by Sec1/Munc18-like (SM) proteins that function as SNARE chaperones but the mechanisms by which SMs regulate SNARE complex formation is not well understood. A crystal structure of the SM protein Vps33 bound to its R-SNARE, Nyv1, and to its Qa-SNARE, Vam3 was recently determined (A.E. Stanton et al., unpublished). However, the crystallized complex lacked a region of Vam3, the so-called linker, that is thought to be functionally important. Furthermore, the electron density corresponding to Nyv1 and Vam3 was weak, preventing confident side chain assignment. This thesis reports additional biochemical and structural studies of Vps33-Nyv1-Vam3 complexes. First, we combined site-directed mutagenesis and a novel pull-down assay to test the contributions of individual Nyv1 residues. Four residues were found to contribute to the binding interaction between Vps33 and Nyv1, corroborating these Nyv1 residue assignments in the existing Vps33-Nyv1-Vam3 crystal structure. Second, we attempted to obtain new crystal structures that included the Vam3 linker region, both to observe its conformation and in hopes that the crystals might diffract to higher resolution. We used three different Vam3 constructs in crystallization trials optimizing three sets of conditions. Diffraction data were collected for crystals grown in two of these conditions; however, no additional resolution was achieved. Together, these studies corroborate four Nyv1 residue assignments in the existing Vps33-Nyv1-Vam3 complex structure, advance knowledge of the Nyv1 residues that play a significant role in the conserved binding interaction between the SM protein and its cognate R-SNARE, and inform future research regarding the conformation of the Qa-SNARE linker region and the potentially conserved role it may play in SNARE complex assembly.