Fig. 4. Mutating four residues around the F^45.52 affect c-Epi affinities in the β₁AR and β₂AR.

a, b F^45.52 (yellow spheres) is conserved in the β₁AR and β₂AR, but is surrounded by differing residues. The different surrounding residues are W199^ECL2, V209^ECL2, K347^6.58, and F359^7.35 in the β₁AR (a) and Y174^ECL2, F194^ECL2, H296^6.58, and Y308^7.35 in the β₂AR (b). c, d Mutating the four residues surrounding F^45.52 reduced c-Epi affinity to the β₂AR and increased c-Epi affinity to the β₁AR, in a β-arrestin recruitment assay (c) and in a competition binding assay (d). Data were given as mean ± SEM of n = 7 (β₁AR (4 mut), β₂AR (4 mut), arrestin), n = 15 (β₁AR, arrestin), and n = 21 (β₂AR, arrestin), n = 6 (β₁AR, β₂AR, β₁AR (4 mut), β₂AR (4 mut),DHA binding) independent experiments. Source data are provided as a Source data file. e F^45.52 has a slightly different conformation in the β₂AR-c-Epi, β₂AR-Epi, and β₁AR-Epi structures, and as a result, the distance between V^3.36 and F^45.52 is longer in the β₂AR-c-Epi structure than in the β₂AR-Epi structure, while the distance is the shortest in the β₁AR-Epi structure. f The distribution for the distance between the Cα atom of V^3.36 and the Cζ atom of F^45.52 in MD simulations are displayed for the different simulation conditions. The distribution is shown in light blue for β₁AR-Epi, dark blue for β₁AR-c-Epi, light green for β₂AR-Epi, dark green for β₂AR-c-Epi and purple for β₁AR_4mut-c-Epi. Cζ of F^45.52 is located deeper in the pocket for β₁AR-Epi compared to β₂AR-Epi, and β₁AR-c-Epi shows a similar distribution as β₁AR-Epi. The deeper position in β₁AR cannot accommodate for c-Epi and leads to a distortion of the F^45.52 conformation and, therefore, to higher distances. For β₁AR_4mut-c-Epi, distances at a β₂AR-like level are observed, suggesting that the β₂AR-like surrounding of F^45.52 leads to conformations of F^45.52 that are located less deep in the pocket as for β₁AR-Epi.