Extended Data Figure 7: Binding of NB80 to agonist-activated TS-β₁AR(A227Y/L343Y).

a, Sequential addition of isoprenaline (1 mM, blue), NB80 (120 μM, red), and cyanopindolol (1 mM, green) to TS-β₁AR(A227Y/L343Y) (120 μM) followed by ¹H–¹⁵N TROSY spectra. Addition of an equimolar amount of NB80 changes the spectrum drastically. Except for few resonances (62, 89, 102, 103, 172, 280 and 326), the assignments are not transferable from the isoprenaline complex. The addition of cyanopindolol to this mixture apparently replaces isoprenaline in the receptor and drives the spectrum to the purely cyanopindolol-bound form. These results indicate that TS-β₁AR(A227Y/L343Y) can be activated, but that the fully active state requires stabilization with a G protein-like partner at the intracellular site. b, Changes in isoprenaline affinity of β₁AR induced by NB80 binding. The isoprenaline affinity was assayed for TS-β₁AR (orange), TS-β₁AR(A227Y/L343Y) (green), and the truncated native turkey β₁AR receptor (tβtrunc, blue)¹⁷ by radio-ligand competition using ³H-dihydroalprenolol in the presence of saturating amounts of NB80 (10 μM, squares) or its absence (circles). For TS-β₁AR(A227Y/L343Y) and tβtrunc the apparent affinity increases by two log units in the presence of NB80, whereas no change occurs for TS-β₁AR. Data points are shown as mean and standard deviation of three independent experiments for each condition. c, Combined ¹H and ¹⁵N chemical shift changes (Δδ = (Δδ_1H²/2 + Δδ_15N²/50)^1/2) between the isoprenaline-TS-β₁AR(A227Y/L343Y) and the NB80-isoprenaline-TS-β₁AR(A227Y/L343Y) complexes as a function of residue number. Except for the few assigned peaks in the NB80-isoprenaline-TS-β₁AR(A227Y/L343Y) complex, chemical shift deviations are calculated as the minimal difference from the isoprenaline peak position to the nearest peak position in the isoprenaline–NB80 complex. Note that these minimal chemical shift deviations present a lower boundary for the deviations that could be obtained from a full assignment of the isoprenaline–NB80 complex. Peak deviations are colour-coded in red (≥0.2 p.p.m.), orange (0.2–0.05 p.p.m.), blue (<0.05 p.p.m.), and grey (ambiguity from peak overlap). d, Representation of the chemical shift response to nanobody binding given in c on the structure of β₁AR. Valines in TM1 and TM7 do not show a large chemical shift response. Due to overlap the response for V280(6.25) in TM6 is unclear. Valines in TM2 show a strong response around the sodium binding region near residue D87(2.50), but valines at its extracellular side are not responsive. In general, valine resonances at the extracellular sides of TM3–TM6 are severely shifted. This indicates that the NB80 binding at the intracellular side has a long-range effect on the extracellular side near the ligand binding site.