Figure 6. Gα_T Helical domain conformations in the Nb35*-free Rho-G_T complex.

(A) Orthogonal views of the complex with the rGα_T helical domain (αHD) in the three observed distinct conformations (colored in orange, green and purple respectively) compared to the conformations of the helical domain observed in the β₂AR-G_S complex (PDB: 3SN6, dark blue) and GDP-bound G_T (PDB: 1GOT, yellow) crystal structures. The comparison is based on the Gα Ras domain alignment. Rho (red), Gβ_lγ₁ (grey) and rGα_T Ras domain (cyan) are from the 3.9 Å Nb35*-free complex structure. (B-D) Interactions between αHD and Gβ₁ revealed by docking the αHD crystal structure (PDB: 1GOT) and the Gβ₁ structure from the 3.9 Å Nb35*-free Rho-G_T complex into the three 4.5 Å Nb35*-free complex cryo-EM maps. Gβ₁ is colored in cyan. αHD is colored in orange, green and purple, respectively, in three different conformers. Residues from Gβ₁ and αHD, that are positioned to form contacts, are shown as blue and red spheres respectively. (E) Trp fluorescence assay monitoring Rho-catalyzed nucleotide exchange in wild-type rGα_T (red), the rGα_T helical domain mutants changing αB-αC loop residues (E111A, E112A) (blue), αD-αE loop residues (E141A, Q143A) (green) or residues from both αB-αC loop and αD-αE loop (D108A, E111A, E112A, E141A, Q143A) (magenta). (F) t_1/2 (time needed for 50% of the fluorescence change to occur) of Rho-catalyzed nucleotide exchange in rGα_T wild-type and mutants from Figure 6E, and the rGα_T helical domain mutants (fluorescence curves shown in Figure S7E) changing αA helix residues (Q75A, R82A, T86A) (cyan), residues from both αA helix and αB-αC loop (Q75A, R82A, T86A, D108A, E111A, E112A) (light blue), or residues from both αA helix and αD-αE loop (Q75A, R82A, T86A, E141A) (orange). Data represents mean ± SEM (n=3). (G) Trp fluorescence assay monitoring response to the addition of AlF₄⁻ in both wild-type and mutant Gα_T (color scheme the same as in Figure 6E).

See also Figures S4, S7 and Tables S2 and S3.