Figure 3.

Experimental ligand competition between α-ionones, and β-ionone in comparison with predicted binding affinities from Docking and free energy calculations. (a) Experimental competition assay². The β-ionone induced Ca²⁺ responses of HEK293 cells transiently expressing PSGR is inhibited by the co-application of α-ionone in a dose-dependent manner. Co-application of increasing concentrations of α-ionone caused an increased reduction of the number of cells responding to 50 μM β-ionone. Shown are relative numbers of cells responding to 50 μM β-ionone in the presence of varying concentrations of a racemic mix of α-ionones. Odorants were applied for 20 s. The data are the mean of 10 independent experiments for every α-ionone concentration tested, each with 600 to 1600 cells. Significance was calculated by Student’s t-test for each sample group referring to cell responses to β-ionone (50 µM). Error bars represent SEM, (*p < 0.05, **p < 0.01 and ***p < 0.001). α-ionone blocks cellular responses to β-ionone, with an affinity that is seemingly twofold higher than the one of β-ionone. (b) Best docking poses of (R)-, (S)- α-ionone, and β-ionone found in the initial ligand-free inactive PSGR model with respective calculated ∆G_bind values. Protein backbone in grey, β-ionone in orange sticks, (R)- α-ionone as cyan sticks, (S)- α-ionone as blue sticks. Selected surrounding protein residues in grey sticks. Helices numbered in roman numbers. β-ionone and (R)- α-ionone bind in a very similar fashion with the head groups oriented towards Asp190^5.47; the detailed binding mode however differs, as (R)- α-ionone presents its keto group oxygen atom to Asp190^5.47, while β-ionone presents the terminal methyl group to Asp190^5.47. (S)- α-ionone binds with the head group oriented towards Lys185^5.42. Both α-ionone enantiomers bind with a slightly higher affinity than β-ionone, which is in line with α-ionone being a competitive antagonist for β-ionone. (c) Results from free energy calculations (see Table SIII). Both α-ionone stereoisomers exhibit a clearly separated affinity for the active and the inactive receptor conformation, with the affinity for the inactive conformation being significantly higher than the one for the active conformation. β-ionone exhibits an affinity for the receptor, which is comparable to the one of α-ionones for the inactive receptor, but within the range of its standard deviation the same for the active and the inactive receptor.