Extended Data Fig. 5: Intramolecular interactions of the Stachel sequence and effects of corresponding mutations.
From: Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4
a, b, Schematic representation of the intramolecular interactions of the Stachel sequence of ADGRG2-β (a) and ADGRG4-β (b). Note that, in ADGRG2-β, the side chains of Y7795.36 and T6211.40 of the upper rim form hydrogen bonds with the main chain of the Stachel sequence, and an intersegment salt bridge was formed by the side chains of D606 and R609. The Stachel sequence in ADGRG4 also anchor with T27471.43. These interactions may help to define the orientation of the peptide and stabilize the overall configuration of the Stachel sequence. Consistent with these observations, elimination of the side chains of T621, Y779 in ADGRG2-β, or T2747 in ADGRG4-β by Ala substitutions significantly impaired the constitutive activity of ADGRG2-β or ADGRG4-β (refer to Supplementary Fig. 5a–f), respectively. The dashed lines with distinct colours represent different interactions (red: charge-charge; green: cation-π; blue: hydrogen bond; orange: polar interaction; black: hydrophobic; pink: π-π). c, Effects of the T599 mutations of ADGRG2-β on intracellular cAMP accumulation at different receptor expression levels. Wild-type ADGRG2-β was used as the control. The intracellular cAMP levels of wild-type ADGRG2-β and its mutants were measured at indicated expression levels as shown in d. Values are the mean ± s.e.m. of three independent experiments for wild-type ADGRG2-β and its mutants (n = 3). Statistical differences between wild-type ADGRG2-β and its mutants were determined by two-sided one-way ANOVA with Tukey’s test, ***P < 0.001; **P < 0.01; n.s., no significant difference. (P = 0.8577, 0.0036, 0.0011, 0.0083, 0.0004, 0.0006 from left to right for T599ss01V; P = 0.3887, 0.0055, 0.0018, 0.0004, <0.0001, 0.0002 from left to right for T599ss01I). d, Similar expression levels of wild-type ADGRG2-β and its T599 mutants are shown by ELISA experiments when HEK293 cells were transiently transfected with indicated amounts of plasmids. Values are the mean ± s.e.m. of three independent experiments for wild-type ADGRG4-β and its mutants (n = 3). Comparisons between the wild-type ADGRG2-β and its mutants were determined by two-sided one-way ANOVA with Tukey’s test. n.s., no significant difference. (P = 0.9839, 0.9763, 0.2183, 0.1553, 0.8098, 0.5447 from left to right for T599ss01V; P = 0.3190, 0.9636, 0.0509, 0.2852, 0.2241, 0.9469 from left to right for T599ss01I). e, RMSD analysis of 200 ns MD simulation of ADGRG2-β, ADGRG2-β-T599V, ADGRG2-β-T599I. Each MD simulation was performed three times (marked with red, dark green and dark blue, respectively). f, The best estimate residue energy contributions of T599V or T599I with 7TM bundle of ADGRG2-β was presented as ΔΔG ((ΔΔG = ΔG Wild-Type - ΔG T599V or T599I)). Values are the mean ± s.e.m. of three independent experiments (n = 3). ***P < 0.001, ADGRG2-β wild-type (WT) and its T599V or T599I mutants were compared. (P < 0.0001, <0.0001 from left to right).
