Fig 1.
Role of the α5 helix in the interaction between R* and G that leads to nucleotide exchange.
From left to right. (A) Membrane anchored GGDP with an unstructured α5 C-terminus encounters R* with a partially unstructured cytoplasmic crevice. (B) The intermediate R*•GGDP complex is formed through mutual structuring of the α5 C-terminus and the R* cytoplasmic crevice. The α5 helix has not yet rotated compared to unbound GGDP. (C) Rotation of α5 lowers the energy barrier separating R*•GGDP from nucleotide free R*•Gempty resulting in GDP release. (D) Uptake of GTP and dissociation of GGTP completes the nucleotide exchange reaction.
Fig 2.
Comparison of the β2AR*•GsGDP model (left panel) and the β2AR*•Gsempty X-ray structure (right panel).
The figure illustrates potential hydrogen bonds to residues within the cytoplasmic crevice (cyan cartoon) from (A, B) the C-terminal reverse turn and (C, D) the N-terminus of GsαCT. (A, C) shows the intermediate position obtained from flexible docking of 15-mer GsαCT (yellow cartoon) and (B, D) the position in the nucleotide free complex (magenta cartoon), respectively. Residue labels from β2AR* are colored in black, from GsαCT in red. Potential hydrogen bonds are denoted as black dashed lines. (E) Complete model of the β2AR*•GsGDP intermediate compared to (F) the β2AR*•Gsempty X-ray structure (PDB entry 3SN6). R*•GGDP was obtained by superposition of GsαGTPγS (PDB entry 1AZT) with the intermediate β2AR*•GsαCT complex by common backbone atoms. Black arrows indicate the rotation of α5.
Fig 3.
Switch of GsαCT (left) and GtαCT (right) at the R* interface observed in MD simulations.
Background figure: GsαCT switches within the cytoplasmic crevice of β2AR* from the intermediate (red) to the nucleotide free position (blue). The transition is schematically indicated by semi-transparent colored cartoons. GsαCT is rotated around its helix axis (red and blue arrows) by about 60°, which eventually triggers GDP release from the nucleotide binding pocket of the Gs holoprotein (gray, flat shaded). In addition a tilt motion of GsαCT parallel to the membrane plane is observed. The surface of the receptor (gray) is cut at the position of R3.50 (orange patch) located at the floor of the cytoplasmic crevice. TM helices are shown as cylinders. For clarity, H8 and H6 of β2AR* are omitted. The panel in the foreground shows rotation of (A) GsαCT or (B) GtαCT around its helix axis; backbone-RMSD of (C) GsαCT or (D) GtαCT relative to the position in the X-ray structure; distance between (E) the center of the phenyl ring of Y391 of GsαCT and R1313.50 or (F) between the carbonyl oxygen of C347 of GtαCT and R1353.50. Gray bars indicate the range of mobility of GαCT in MD simulations of the X-ray structures of (left) holo β2AR*•Gsempty (taken from ref. [25]) or (right) RhR*•GtαCT (see Figs B, C and E in S1 File; see Methods section). The mobility of switched GsαCT (after about 100 ns) is only slightly increased, when compared to the mobility of the corresponding section in β2AR*•Gsempty (grey). The time series data are drawn on top of the raw data as a running average. The plots are linear for the first 10 ns and logarithmic for the remaining time (gray dashed lines). The four representative simulations (black, red, blue, green) of 11-mer GsαCT (Panel A of Fig N in S1 File, simulations 8, 9, 21 and 23) and of 11-mer GtαCT (Panel B of Fig N in S1 File, simulations 9, 16, 21 and 30) were picked from 8 and 10 simulations were a helix-switch was observed (Fig N in S1 File).