Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structural basis of frizzled receptor activation upon ligand binding in non-canonical Wnt signalling: - (#346)

Susovan Das 1 , Hariprasad Venugopal 2 , Katrina Black 1 , Tin Nguyen 1 , Lilian Wong 1 , Mahmuda Mun 3 , David Thal 3 , Alisa Glukhova 1
  1. Structural Biology Division, Walter and Eliza Hall Institute of Medical Research (WEHI), Parkville, VICTORIA, Australia
  2. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

FZD receptors tightly regulate different biological processes and are involved in different cancers [1]. Wingless/Int1 (WNT) family proteins act as ligands for FZDs. They initiate distinct signalling pathways broadly separated into the "canonical" or beta-catenin-dependent and the "non-canonical" pathways [2], [3]. At least some of the non-canonical pathways are mediated via FZD coupling to heterotrimeric G proteins. As all class F G protein-coupled receptors, FZD have a long extracellular N-terminal cysteine-rich domain (CRD) and a seven-transmembrane domain (7TM). While individual structures of CRD-Wnt complexes and individual 7TM have been determined, there is no structural information on how WNTs bind to and activate full-length FZDs or what conformation CRD adopts upon receptor activation. SAG1.3 is a Smoothened receptor agonist that also activates FZD receptors[4]. Due to challenges with WNTs' hydrophobic nature and stability, we decided to use SAG1.3 to examine the molecular details of FZD activation.

We co-expressed FZD7-mGs complex in insect cells and purified it in the presence of SAG1.3. The purity and composition of the complex were confirmed by the Coomassie-stained gel and the Western blot. Complex formation was further confirmed by negative-stain electron microscopy. The high-resolution dataset was collected using a Titan Krios microscope and yielded a 2.6Å map. Unfortunately, no SAG1.3 or CRD were observed, most likely due to the low affinity of SAG1.3 and the flexibility of the FZD CRD domain. Currently, we are investigating whether other G proteins might form more stable complexes with FZD7 and SAG1.3 (by looking at FZD7-mGi complexes) or whether CRD flexibility might be reduced in other FZD receptors (FZD5-mGq complexes).

 

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  2. E. Vincan, Ed., Wnt Signaling, vol. 468. Totowa, NJ: Humana Press, 2008. doi: 10.1007/978-1-59745-249-6.
  3. B. T. MacDonald, K. Tamai, and X. He, “Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases,” Developmental Cell, vol. 17, no. 1. pp. 9–26, Jul. 21, 2009. doi: 10.1016/j.devcel.2009.06.016.
  4. P. Kozielewicz et al., “Structural insight into small molecule action on Frizzleds,” Nat Commun, vol. 11, no. 1, Dec. 2020, doi: 10.1038/s41467-019-14149-3.