Role of structural dynamics at the receptor G protein interface for signal transduction

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket

A Ligand Channel through the G Protein Coupled Receptor Opsin

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 Å and is between 11.6 and 3.2 Å wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal β-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90° elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B

Position of transmembrane helix 6 determines receptor G protein coupling specificity

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαβγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active β2-adrenoceptor (β2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαβγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed β2AR*. The slim GiαCT stabilizes a β2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of β2AR* toward Gi or Gs

Role of the α5 helix in the interaction between R* and G that leads to nucleotide exchange.

<p>From left to right. (A) Membrane anchored G^GDP with an unstructured α5 C-terminus encounters R* with a partially unstructured cytoplasmic crevice. (B) The intermediate R*•G^GDP 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 G^GDP. (C) Rotation of α5 lowers the energy barrier separating R*•G^GDP from nucleotide free R*•G^empty resulting in GDP release. (D) Uptake of GTP and dissociation of G^GTP completes the nucleotide exchange reaction.</p

Comparison of the β₂AR*•Gs^GDP model (left panel) and the β₂AR*•Gs^empty X-ray structure (right panel).

<p>The figure illustrates potential hydrogen bonds to residues within the cytoplasmic crevice (cyan cartoon) from <b>(A, B)</b> the C-terminal reverse turn and <b>(C, D)</b> the N-terminus of GsαCT. <b>(A, C)</b> shows the intermediate position obtained from flexible docking of 15-mer GsαCT (yellow cartoon) and <b>(B, D)</b> 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. <b>(E)</b> Complete model of the β₂AR*•Gs^GDP intermediate compared to <b>(F)</b> the β₂AR*•Gs^empty X-ray structure (PDB entry 3SN6). R*•G^GDP was obtained by superposition of Gsα^GTPγS (PDB entry 1AZT) with the intermediate β₂AR*•GsαCT complex by common backbone atoms. Black arrows indicate the rotation of α5.</p

Switch of GsαCT (left) and GtαCT (right) at the R* interface observed in MD simulations.

<p>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 R^3.50 (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 <b>(A)</b> GsαCT or <b>(B)</b> GtαCT around its helix axis; backbone-RMSD of <b>(C)</b> GsαCT or <b>(D)</b> GtαCT relative to the position in the X-ray structure; distance between <b>(E)</b> the center of the phenyl ring of Y391 of GsαCT and R131^3.50 or <b>(F)</b> between the carbonyl oxygen of C347 of GtαCT and R135^3.50. Gray bars indicate the range of mobility of GαCT in MD simulations of the X-ray structures of (left) holo β₂AR*•Gs^empty (taken from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.ref025" target="_blank">25</a>]) or (right) RhR*•GtαCT (see Figs B, C and E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#sec010" target="_blank">Methods</a> 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*•Gs^empty (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>, simulations 8, 9, 21 and 23) and of 11-mer GtαCT (Panel B of Fig N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>, simulations 9, 16, 21 and 30) were picked from 8 and 10 simulations were a helix-switch was observed (Fig N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>).</p