Fig. 4: Mutagenesis experiments validate computationally derived activation mechanism.

At the mutated G3.49E receptor, AP8 acts as an inverse agonist. FFAR1 activity was monitored in IP1 accumulation assays in HEK293 cells expressing WT or G3.49E mutant receptors treated with a AP8 or b MK-8666. Data is plotted as the % of WT receptor basal activity (cells treated with 1% DMSO), where data points are mean ± S.E.M. from N = 3 biologically independent experiments and n = 2 technical replicates. Dose-response curves were fit to a standard 4-parameter non-linear regression model. Images at right show simulation frames in color and starting structure as a black outline; AP8 is displaced from its WT binding pose by G3.49E, likely due to the repulsion of the two nearby carboxylates. c In simulations, AP8 destabilizes the PR ICL2 state at the G3.49E receptor, the opposite of its behavior at the WT receptor. Data presented as mean with 68% CI (P = 0.031 WT –AP8 vs. +AP8, P = 0.027 WT –AP8 vs. G3.49E –AP8, two-sided MWU test, from left N = 5, 5, 10, 10 independent simulations for each condition). d Basal receptor activity in the IP1 accumulation assay, normalized by receptor surface expression, is plotted at right for different mutants at the 3.49 position. Only G3.49E leads to an increase in basal receptor activity relative to WT. At left, a snapshot of the simulation of the G3.49E receptor in complex with Gq shows the glutamate sidechain can mimic the interactions of the AP8 carboxylate. Data are expressed as the mean ± S.E.M. from a single fit to grouped data from N biologically independent experiments (from left, N = 3, 3, 2, 3, 2) and n = 2 technical replicates.