Extended Data Fig. 6: Characterization of phospholipid in GPR156.
a, Comparative lipidomics analysis of endogenous lipids bound to purified recombinant GPR156. PC and PE are enrichment in GPR156 fraction relative to total cellular lysate. Bars show mean ± standard deviation from three independent experiments (dots). b–c, GPR156 activation as measured by GTPase-Glo assay for GPR156-Gi peptidisc containing PE (b) or GPR156 in LMNG (c). A peptidisc containing GPRC5D-Gi and PE was incubated with PG as a control. Lower levels of residual GTP indicate higher level of G-protein activity. Data in (b) and (c) were mean ± SEM from three independent experiments, performed in technical duplicate. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test. d‒f, Comparison of molecular dynamics simulation of GPR156 Go-coupled complexes with PG versus PC. d, Root-mean-square-fluctuation (r.m.s.f.) calculated of each subunit in the complexes; shading refers to 95 % confidence interval (n = 5). e–f, Distribution of the closest distances between the sidechain of R2796.57 and the phospholipids against the closest distances between the backbone of C216ECL2 and the phospholipids on the nGC protomer (e) and on the GC protomer (f). The shading refers to density estimated with a multivariate gaussian kernel; the marginal distribution (by count) is shown on the sidebar. The black points in the background is the datapoints collected every 0.5 ns. All distances were calculated using only the heavy atoms. The horizontal and vertical dotted line refers to 3.5 Å. g, Mutational effect on the F215ECL2 adjacent to the phospholipid head in G-protein activation, measured by BRET2 assay. Data were mean ± SEM from four independent experiments, performed in technical triplicate. Statistical differences were analyzed by two-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). h‒i, Comparison of PC- and PG-bound GPR156 with representative snapshots from MD simulations of GPR156. Close-up views of a phospholipid-binding site in the simulations of PC-bound (h) and PG-bound GPR156 (i). nGC protomer and GC protomer are shown on top and bottom, respectively. Three representative snapshots (970, 985, and 1000 ns) of a simulation trajectory are displayed.
