Fig. 6: Ligand-induced chemical shift changes at the extracellular end of TM6.

a Selected region from ¹H-¹⁵N TROSYs showing the variation of the V314^{6.59 1}H-¹⁵N resonances in the apo form and in orthosteric binary and ternary isoprenaline•Nb80 complexes of YY-β₁AR at 21 T and 304 K. Due to limited stability, the apo form of YY-β₁AR was recorded at 294 K. Spectra are color-coded and marked according to the ligand. Due to exchange broadening, the resonance marked by ‘Iso^s’ was only observed at 14 T. It corresponds to the second, minor conformation of the YY-β₁AR•isoprenaline complex and coincides with the resonance of V314^6.59 resonance in the ternary isoprenaline•Nb80 complex (orange). Unrelated resonances are marked by ‘x’. A dashed line visualizes the approximate linear correlation between ¹H and ¹⁵N chemical shifts of V314^6.59 in various receptor states. b Hydrogen bond distances d_HiOi-4 and d_HiOi-3 for residues 6.54, 6.59 and 6.60 in β₂AR (square) and β₁AR (circle) from Phenix ensemble calculations for binary orthosteric ligand complexes (2Y03, 2RH1, 2VT4 chain A and B; blue) and ternary agonist complexes (4LDL, 6H7J; orange). Source data are provided as a Source Data file. c Ensemble backbone structures ranging from residue 6.50–6.59 derived by the Phenix ensemble calculations for β₁AR•isoprenaline (2Y03, cyan) and β₁AR•isoprenaline•Nb80 (6H7J, yellow). The 3₁₀ hydrogen bond H^6.59•••O^6.56 in the agonist-bound structure is shown as a dotted line. d Evidence for the TM6 pivoting in response to nanobody binding in the crystal structures of β₁AR. Structures of the binary complex with the agonist isoprenaline (2Y03) and of the ternary complex with agonist isoprenaline and Nb80 (6H7J) were aligned on the central region of the unaffected helices TM1-4,7 (shown in gray for 2Y03). TM5 and TM6 are depicted in cyan for the binary complex and in yellow for the ternary complex. The V314 N atoms are depicted as spheres, nanobody Nb80 as an orange ribbon. e Ligand-binding pocket of the binary and ternary complex β₁AR structures as shown in d. Residues V314^6.59, R205^5.37 and ligands are depicted in stick representation. Van der Waals spheres are shown for V314^6.59 and R205^5.37 in the ternary complex to visualize their contact.