Structural analysis of the HOPS tethering complex

• Applicants: Professor Dr. Arne Möller; Professor Dr. Christian Ungermann
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2007
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 37802001

Tethering complexes are essential in organelle biogenesis and integral parts of the cellular fusion machinery. They bind to vesicles and organelles, which enables their association, and assemble SNAREs to promote fusion. HOPS is the only tethering complex, for which a functional assay exists, yet its role in fusion remains speculative in the absence of detailed structural insights. High-resolution structural data would deliver the mechanistical insights that are missing to unravel the role of tethering complexes on macromolecular scale. Given the high flexibility of such complexes, all attempts to provide structural information at high resolution have failed. Here, we combine improved purification techniques and liposome-based functional assays (Ungermann lab) with novel structural approaches in EM (Moeller lab) to resolve the high-resolution structure of HOPS. In Aim 1, we will focus on the biochemical optimization of HOPS to enable subsequent high-resolution analysis by cryo-EM. In Aim 2, we will address how HOPS interacts with its ligands (Ypt7, SNAREs, AP-3) by structural analyses. In vitro reconstitution approaches such as tethering and fusion assays will be accompanied by (i) mutant complementation in yeast, and (ii) analysis of mutant HOPS complexes with impaired Rab- and SNARE-binding abilities to validate the conclusions and ideas drawn from our structural analyses. In Aim 3, we will analyze HOPS conformations on and between membranes using liposomes and nanodiscs to finally uncover how HOPS functions at membranes.

• DFG Programme: Research Grants