Fig. 5. Schematic overview of β₁AR ligand activation and ternary complex formation.

Cartoon cross sections spanned by TM6, TM7 and H8 with the TM7 ^TETC344^7.54 probes shown as yellow spheres, adjacent to Y343^7.53 of the NPxxY motif. The receptor exists in an equilibrium of inactive states (I_1,2) and a pre-active state (A), with the latter populated in growing amounts with increasing efficacy of the bound ligand. Nanobody Nb6B9 addition leads to the formation of a fully active ternary complex (A^G+) in amounts proportional to the ligand efficacy. Nanobody binding also occurs with the inactive form of the receptor, resulting in the formation of (A^G−). The latter can be considered as a pre-coupled inactive form, with inactive and active ternary complexes in slow exchange with each other. The binding interface in the (A^G−) complex is shown faded, emphasising that Nb6B9 has not yet fully engaged the epitope characteristic of the active receptor state. The (I₁) ⇌ (I₂) interchange takes place on a μs-to-ms timescale, while the (I₂) ⇌ (A) interchange as well as the (A^G−) ⇌ (A^G+) exchange occurs on a slower sub-second timescale. Exchange rates, where measured, are indicated below the equilibrium arrows. In the ternary state (A^G+) the cytoplasmic region of TM6 is rigid while TM7 remains dynamic on the μs-to-ms timescale, implied by TM7 showing partly blurred. In (A^G+) the conformation of TM7 in the vicinity of the NPxxY motif reveals agonist-dependent conformational differences, emphasised by TM7 showing in different colours in the enlarged region marked with (*). The grey slider below each receptor cartoon approximates the relative hydrophobic/hydrophilic extent of the TM7 ¹⁹F NMR probe surrounding in that particular state. Lipid bilayer hydrophobic regions are shown in light grey. Dark grey areas indicate transmembrane regions of the receptor with residues rich in hydrophobic side chains. These form hydrophobic gates above and below the NPxxY region (e.g. in I₁ and I₂) that shield the receptor interior against bulk water access. Blue dots on a grey background (e.g. in I₁, I₂ and A^G−) indicate ordered internal water molecules, separated from the bulk water through the hydrophobic gates (dark grey). Conformational changes upon activation disrupt the two hydrophobic side chain layers, resulting in the gradual opening of a continuous internal water pathway with cytoplasmic influx of bulk water, as indicated by the speckled grey/blue area between TM6 and TM7 in (A) and (A^G+).