Supplementary Figure 5: Comparison of the structures of wt PTH and ePTH bound to PTH1R and the role of glycosylation at N161 on PTH binding.
a, Overlay of the crystal structures of the ECDs of PTH1R-ePTH (purple and orange) and of the isolated ECD in complex with wt PTH(1–34) (PDB 3C4M, gray) with ligand residues shown in stick representation. Residues in ePTH differing from wt PTH are highlighted in green. b, Superposition of ePTH with the crystal structure of wt PTH(1–34) (PDB 1ET1, gray). c, ePTH with the Fo– Fc omit density map contoured at 2.5σ. d, ECD of PTH1R with glycosylation (gray) resolved at residue N161 in close proximity to ePTH (orange) shown in sticks. The hydrogen bonds (dashed blue lines) between the amidated peptide C terminus of Y34 and T163 of PTH1R rationalize the reported increase in binding affinity of C-terminal amides over carboxylic acids in PTH(1–34) peptides (Proceedings of the Fifth Parathyroid Conf. 33–39, 1975). The 2Fo – Fc electron density in gray mesh is contoured at 1.0σ. e, Binding of PTH-HL647 to HEK293T cells expressing wt PTH1R and the mutant PTH1R_N161D in the presence of different concentrations of unlabeled PTH peptide. Data are shown as mean values ± s.e.m. from n = 7 independent experiments performed in duplicate. The IC50 values for wt and PTH1R_N161D are listed in Supplementary Table 2.
