Structural analysis of Wnt signalling pathways

Thesis

Wnt signalling through its principal receptor Frizzled (Fzd) is one of the key determinants of metazoan development. Fzd is a member protein of the G-protein coupled receptor family, yet despite a wealth of functional information available on various aspects of its role within cells and during development, the molecular mechanism for Wnt-activated Fzd signalling has remained unknown. To shed light on this question, this project pursued a cryo-EM approach to obtain structures of different Fzd signalling complexes. To facilitate the assembly of complexes containing transmembrane regions of several proteins, as well as their subsequent examination by cryo-EM, I also explored the reconstitution of Fzd within two detergent-free systems, nanodiscs and SMALPs. Both systems posed unique challenges for obtaining well-behaved samples, ultimately proving to be unsuitable for sample examination by cryo-EM. I then returned to detergent systems, focusing on the assembly of Fzd4 in complex with an atypical ligand Norrin and bound Fab fragments, which, after an extensive sample optimization strategy, appeared to be too flexible for structure determination. This may support the theory that instead of functioning as a classic GPCR with defined intrahelical movements and a rigid structure upon activation, Fzd acts as a passive scaffold that brings different intra- and extra-cellular components together to result in signalling.

In parallel to this project, I successfully determined the structures of several small molecule inhibitors in complex with the Wnt deacetylase Notum via X-ray crystallography. This led to the identification of several key determinants controlling the affinity of molecules binding to the Notum catalytic site, thus aiding further drug development targeting this protein.

Authors: Ruza, RR

• DOI: 10.5287/ora-bedmx06bz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Wnt; Notum; Norrin; Frizzled
• Subjects: Biochemistry; Receptor-ligand complexes; Membrane proteins; Developmental biology