Structure of the HOPS complex and its interactions with SNARE proteins
Biochemistry
Final Report Abstract
Eukaryotic cells rely on vesicle trafficking for growth, differentiation, signaling and many other crucial cellular functions. Cargoes are delivered by fusion of the vesicles with the target organelle, which is highly specific and driven by the assembly of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). For membrane fusion, SNAREs collaborate with other trafficking co-factors including Sec1/Munc18 (SM) proteins, Rab GTPases, and membrane tethering factors. Multisubunit tethering complexes (MTCs) are thought to be the major orchestrators of vesicle tethering and fusion. Consistent with this hypothesis, MTCs interact with SNAREs and most of the other trafficking co-factors, but the underlying molecular mechanisms are poorly understood. The homotypic fusion and vacuolar protein sorting (HOPS) complex, is the essential MTC for the fusion of late endosomes. We proposed to determine its structure and to investigate its functional interactions with SNARE proteins. We established the recombinant expression of individual HOPS subunits from Chaetomium thermophilum in E. coli as well as of the complete HOPS complex in insect cells and optimized the respective purification strategies. Negative-stain electron microscopy (EM) experiments indicate that the complex is unstable and displays various conformational states. Initial cryo-EM studies suggest that the complex dissociates during grid preparation. Therefore, we are currently optimizing grid preparation conditions to obtain intact HOPS particles for EM analysis. Concurrently, we are attempting to determine structures of HOPS subunits and subcomplexes by X-ray crystallography. This already resulted in the structure of the N-terminal β-propeller domain of Vps11 at a resolution of 2.3 Å. Recent data from our lab suggested that the SM protein and HOPS subunit Vps33 promotes membrane fusion by binding to SNARE proteins from the two opposing membranes via their SNARE motifs, thereby templating the SNAREs for assembly into a fusogenic four-helix bundle. Therefore, we examined the role of SM proteins in SNARE complex formations using single molecule biophysical experiments. Our recently published data suggest that Vps33 catalyzes the step-wise assembly of the four SNARE motifs into a four-helix bundle via a defined templating pathway and that this function is conserved among SM proteins. Furthermore, we tried to elucidate the role of the regulatory N-terminal domains of SNARE proteins in SNARE complex assembly by biochemical and structural approaches. To date, we could generate a crystal structure of the N-terminal PX domain of C. thermophilum Vam7 at 2.2 Å and identify the region in Vam7 that is important for binding to the HOPS subunit Vps18. The progress in the preparation of the complete C. thermophilum HOPS complex and HOPS subcomplexes as well as the identification of interacting domain between different subunits provides a solid basis for future structural analysis, which will follow the already obtained crystal structures of the HOPS subunit Vps11 and the SNARE Vam7. Using single-molecule force extension experiments, we could show that SM proteins catalyze the formation of a fusogenic SNARE complex via a template complex consisting of the SM protein and the SNARE proteins from opposing membranes. Taken together, the obtained biochemical and biophysical data provide insight into the role of multisubunit tethering complexes and the molecular mechanisms underlying membrane tethering and fusion.
Publications
- (2018) Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association, eLife, 7:e41771
Jiao J, He M, Port SA, Baker RW, Xu Y, Qu H, Xiong Y, Wang Y, Jin H, Eisemann TJ, Hughson FM, Zhang Y
(See online at https://doi.org/10.7554/eLife.41771.001)
