Fig. 3. Differences in the recognition mode of three peptide agonists of MC4R.

a–c Parallel structural comparisons of α-MSH–afamelanotide, α-MSH–bremelanotide and afamelanotide–bremelanotide. The peptide ligands are superimposed upon alignment with MC4R. α-MSH, orange; afamelanotide, magenta; bremelanotide, forest green. d, e Afamelanotide- (d) and bremelanotide-induced cAMP accumulation (e) assays of the residues involved in ligand recognition. Bars represent differences in calculated agonist potency [pEC₅₀] for each mutant relative to the wild-type receptor (WT). Data are colored according to the extent of effect. ^nsP  > 0.01, *P < 0.01, **P < 0.001 and ***P < 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparisons test, compared with the response of WT). See Supplementary information, Tables S8 and S9 for detailed statistical evaluation and receptor expression levels. f Comparison of the binding sites of α-MSH, afamelanotide (AFA) and bremelanotide (BRE) in MC4R. The triangle-labeled residues are crucial for MC4R activation.