Fig. 2: The N-terminal regions of enNTS₁-R213 and enNTS₁ show different conformational dynamics in response to binding agonist and inverse agonist.

¹⁹F NMR spectra of a apo G50tfmF-enNTS₁-R213 and b in complex with saturating concentrations of NT8-13. Complexation with NT8-13 promotes formation of P4 (red) and reduces the population of P3 (orange). Despite being saturated, ligand cannot remove P2 (green). P1 (purple), which is due to cis/trans isomerization of Pro51 (Supplementary Fig. 7) disappears. The blue and cyan line in each spectrum indicate the sum and residuals of the deconvoluted spectrum, respectively. ¹⁹F NMR spectra of c apo G50tfmF-enNTS₁ (residue 213 is Leu) and d in complex with saturating concentrations of NT8-13. Colour scheme is the same as described in (a, b). P3 is more populated in apo G50tfmF-enNTS₁ compared to G50tfmF-enNTS₁-R213. Upon binding to NT8-13, P1 disappears and P4 formation is promoted, which is slightly shifted downfield compared to P4 of G50tfmF-enNTS₁-R213 (b). ¹⁹F NMR spectra of e apo G50tfmF-enNTS₁-R213 and f in complex with inverse agonist SR142948A. Colour scheme is the same as described in (a, b). P3 is increased in population in the complex with inverse agonist, but the signal P4 is not observed compared to spectra in the presence of peptide agonist. P1 is also observed, suggesting that the N-terminal region retains flexibility. The chemical shift and population of each state is summarized in Supplementary Table 1. g Structural models of enNTS₁ where P1/P2 show significant flexibility of the N-terminal region. P4 shows the N-terminal region and ECL2 form a closed conformation upon binding of NT (green). An intermediate state, P3, is formed by loose interaction of ECL2 with the N-terminal region of the receptor. P3 is populated in both apo state and ligand-bound state of the receptor.