Fig. 4: Localization of zinc-binding hotspots and their functional impact on Gs-protein coupling.
a, MS spectrum of the tβ1AR–Nb6B9 complex incubated with isoprenaline, indicates reduced binding of endogenous metal. The complex is highlighted in green and its metal-binding stoichiometry is reduced to first order (denoted as 1×). The peaks assigned to Nb6B9 alone are highlighted in brown. b, MD simulation of the tβ1AR–mini-Gs complex in the presence of zinc revealed two zinc-binding hotspots. The zinc contacts in the orthosteric ligand-binding site (Asp 121, Asn 329 and Tyr 333) are shown in the upper box (labelled 1), and a subset of the zinc contacts at the intracellular interface are highlighted on the receptor (Glu 233, Glu 236, Gln 237, Glu 285 and His 286) and mini-Gs (Asp 381, Gln 384 and Glu 392) in the bottom box (labelled 2). c, The impact of mutations to the zinc contacts on tβ1AR–mini-Gs complex formation and metal binding (orange boxes). Two receptor variants (E233A&E236A and E285A&H286A) and a mini-Gs mutant (E392A) were examined. Representative MS spectra from three independent experiments are shown. The receptor monomer and receptor–mini-Gs complex are shown in blue and orange, respectively. d, The inhibitory effect of zinc-contact mutants on tβ1AR–mini-Gs complex formation (orange bar) and metal binding (blue bar). The percentage inhibition was calculated from the results acquired with wild-type tβ1AR and mini-Gs, shown in the top spectrum of c. The dots represent the individual data points and the bars represent the mean ± s.d. from three independent experiments on the same batch of purified proteins.
