Fig. 5: The allosteric effect of endogenous zinc on the selectivity of mini-Gs.
a, Representative MS spectra of tβ1AR coupling to wild-type (WT) mini-Gs and the E392A mutant in response to isoprenaline at 25 μM. The GDP-bound and GDP-free complexes are highlighted in green and orange, respectively, and the receptor monomer is indicated in blue. The binding stoichiometry of the metal ion is shown in the orange boxes. b, Relative percentages of the stoichiometric distribution of the tβ1AR monomer and tβ1AR–mini-Gs GDP-bound and GDP-free complexes. The results indicate weaker complex formation and slower structural transition to the stable state in mini-Gs E392A. c, Binding stoichiometry of the metal ion in the intermediate and stable complexes of tβ1AR–mini-Gs_WT and tβ1AR–mini-Gs_E392A. The bars in b and c show the mean ± s.d. from three independent experiments, including individual data points, on the same batch of purified proteins. d, Schematic representation of the hypothesized activation mechanism of tβ1AR–Gs complex formation according to molecular dynamic simulations performed on the tβ1AR–Gs H5 peptide and tβ1AR–mini-Gs complexes. The receptor first forms contacts with the Gs protein in a GDP-bound state (i), as an intermediate complex it further transits to a conformation where zinc interacts with both Glu 392Gs and Asp 348β1AR (ii), leading to the formation of a stable complex in a GDP-free state (iii). Snapshots of the MD structures illustrate the spatial orientation of the H5 motif of the Gs protein (orange) and its coordination with Zn2+ in the discrete conformations of the complex.
