Figure 4. In vitro assessment of the functional role of the vestibule and the binding cavity.

a) a snapshot where CBA bound at the vestibular site can form a hydrogen bond with R265^6.59 (blue dashed line) and hydrophobic contacts with L203^5.40 and F266^6.60. In this representation extracellular loops are omitted for image clarity. For each residue near the agonist, normalized dose-response curves of the response of OR51E1 mutants (red) in comparison with the wt (black) to CBA stimulation is shown. The y-axis represents the firefly luminescence normalized to Renilla firefly and normalized to the wt response. b) Normalized dose-response curves (error bars represent SD; n=3) for OR51E1 wt to nonanoic acid (NA), isovaleric acid (IVA), and butyric acid (BA). c) Log(EC50) values for the four agonists. Comparison between the wt and the mutant ORs at the position V255^6.49 and at the vestibular site. ‘nr’ indicates non-responsive. Mutation V255^6.49G, which does not interact with agonists has no statistically significant (ns) effect on the EC50 for any agonist. Mutations at the vestibular site differentially affect potency. A ‘*’ indicates that the 95% EC50 confidence intervals do not intersect (Table S3). The H264^6.58A mutation only affects IVA binding while the F266^6.60A mutation affects all ligands except NA. The L203A^5.40 mutation mostly decreases both CBA and IVA potencies d) Normalized basal activity for all mutant ORs normalized to wt. All mutants basal activity are significantly different than the wt (****P<0.001; ANOVA Dunnett’s multiple comparisons test).