Supplementary Figure 2: Comparison of PTH peptide sequences and in vitro pharmacology of ligands and receptor constructs.

a, Sequence alignment of human PTH (wt PTH) and engineered ePTH. Colored boxes depict sequence changes in comparison to wt PTH, and black boxes denote non-natural amino acids. The topology of PTH1R interaction is indicated on top. Ac5c, aminocyclopentane-1-carboxylic acid; Aib, α-aminoisobutyric acid; Hrg, homoarginine; Nle, norleucine. b, Thermostability assay (CPM) of the crystallized ECD-PTH1R_S-PGS (PTH1R_XTAL) fusion bound to wt PTH(1–34), wt PTHrP(1–36) and ePTH (respective melting temperatures are 58.0 °C, 58.4 °C and 65.0 °C). Data shown are from a representative experiment. c, Binding of PTH-HL₆₄₇ to HEK293T cells expressing wt PTH1R in the presence of different concentrations of unlabeled PTH peptides. d, cAMP accumulation measured in HEK293T cells expressing wt PTH1R. e, IP1 accumulation measured in HEK293T cells expressing wt PTH1R. f,g, Binding of PTH-HL₆₄₇ to HEK293T cells expressing wt PTH1R or the crystallized PTH1R with the PGS fusion (PTH1R_XTAL) in the presence of different concentrations of unlabeled ePTH (f) or wt PTH(1–34) (g). h, cAMP accumulation measured in HEK293T cells expressing wt PTH1R or the crystallized PTH1R with the PGS fusion (PTH1R_XTAL). Data shown in c–h are mean values ± s.e.m. from five (c,f,g), four (d,e) or three (h) independent experiments performed in duplicate. The IC₅₀ values for c, f and g are listed in Supplementary Table 2.