The Class F G protein-coupled receptors (GPCRs) include Smoothened and the ten Frizzled receptors, which are the major cell membrane receptors in the Hedgehog and Wnt signalling pathways respectively and of enormous interest in embryonic development and as therapeutic targets in cancer. These receptors consist of a Frizzled-type cysteine rich ectodomain, to which endogenous agonists bind and a seven-helical transmembrane bundle, similar to that found in other families of GPCRs. Despite their biological importance, our understanding of how signalling is modulated by these receptors remains limited. In particular, the existence of 10 Frizzleds, 19 Wnt ligands and multiple adaptor proteins (including but not limited to three Dishevelled isoforms and multiple G proteins) has precluded comprehensive studies of Wnt signalling; it is known that multiple Wnt signalling pathways exist (canonical, planar cell polarity, and calcium-dependent), but the receptor interactions leading to specific signalling outcomes are only known for a limited set of cases. The existence of such a large number of possible ligand-receptor-adaptor combinations suggests that receptor conformational selection may play a role in the direction of signalling towards specific Wnt pathways; recent crystal structures of Smoothened in the active, G protein-bound conformation provide the opportunity to explore this hypothesis in detail.
In this study, we employ adiabatic biased molecular dynamics and umbrella sampling to investigate structurally the activation of Smoothened and Frizzled-7, in both the native state and bound to activating endogenous ligands – cholesterol and Wnt3a respectively – as well as how the clinically used Smoothened antagonist, vismodegib, alters Smoothened activation. We find that activating endogenous ligands significantly lower the energetic barrier for accessing receptor active states. In particular, we find that Wnt3a strongly disfavours the Frizzled-7 inactive state while stabilising active and metastable receptor states, which could representative states binding different adaptor proteins. Vismodegib strongly prevents Smo activation. Our results illustrate for the first time how endogenous ligands lead to Class F GPCR activation, despite the lack of engagement of the transmembrane region, and demostrate the simulation approach as a robust means of broadly investigating signalling through Class F GPCRs.