WalkMat_v4_RSOB

Zipped archive containing ‘WalkMat’ package of Matlab computer code used to run the stochastic simulations and to integrate the downstream reactions in support of the findings in the publication

Precision vs Flexibility in GPCR signaling

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E­(D)­RY and Yx₇K­(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general

Docking of retinal isomers to docking site I.

<p>Flexible docking of (a–c) 11<i>-cis-</i>retinal and (d–f) all-<i>trans</i>-retinal to site I located between opening A and C2 at the 90° kink of the channel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g004" target="_blank">Figure 4</a>). The crystal structure of Ops* (PDB entry 3CAP) was used and full flexibility for Lys296 side chain was allowed. The most likely conformation of (a) 11<i>-cis-</i>retinal and (d) all-<i>trans</i>-retinal is shown together with the neighbouring residues. The conformation of Lys296 obtained by the docking procedure (orange) is superimposed to the starting conformation (light green). Cluster of docking poses of (b) 11<i>-cis-</i>retinal and (e) all-<i>trans</i>-retinal and (c, f) the respective lists of ranked docking poses at different RMSD cut-off values (different colours identify individual poses). The best scored pose of the finally selected cluster (shaded) is shown with ball and sticks in a, b, d and e.</p

Location of the retinal docking sites.

<p>(a) The docking sites (I, II, III) are restricted to all residues within the radius of 10 Å (circles) from Met44 (I), Tyr268 (II) and Ala269 (III), respectively. Site I is close to opening A and the 90° kink of the channel, site II represents the retinal binding pocket, and site III is close to opening B. (b) Selected final conformations of retinal isomers resulting from the three docking procedures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g005" target="_blank">Figures 5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g006" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g007" target="_blank">7</a> for details). Blue, 11-<i>cis-</i>retinal docked to site I; green, all-<i>trans</i>-retinal docked to site II; and cyan, all-<i>trans</i>-retinal docked to site III. (c) View onto the ligand channel (electrostatic surface potentials as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g001" target="_blank">Figure 1a</a>) with docked 11-<i>cis</i>- (blue) and all-<i>trans</i>-retinal (green, cyan). Parts of the receptor were omitted to visualize the two openings (A and B), the constrictions (C1–C4) and the neighbouring cavity (NC).</p

Structural features of the opsin ligand channel.

<p>Coplanar cut through opsin revealing the channel with opening A, B and constrictions C1-C4 (a, top view). The position of Lys296 is indicated by a yellow dot. All-<i>trans</i>-retinal (green) is docked into the binding pocket (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#s4" target="_blank">Methods</a>). Electrostatic surface potentials were calculated using the program APBS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone.0004382-Baker1" target="_blank">[22]</a> with nonlinear Poisson-Boltzmann equation and contoured at ±20kT/e and negatively and positively charged surface areas in red and blue, respectively (at high kT/e values the contour level is shifted from coloured to grey scale). (b, c) Side-views, with electrostatic surface potentials contoured at ±8kT/e. (d, e) Close-ups of openings A and B, defined by the residues as indicated.</p

Effect of Lys296 conformation on channel constriction.

<p>(a) Clusters (1–3) of calculated Lys296 rotamers. Orange (cluster 2), calculated rotamers of Lys296 as also found in the crystal structure of Ops*-GαCT (PDB entry 3DQB). Light green (cluster 3), calculated conformation of Lys296 as used for skeleton search (see text for details). (b) Superposition of the two most plausible conformers of Lys296 shown with neighbouring residues (distance <5 Å) and with the potential network of hydrogen bonds (dashed lines). (c) View onto the ligand channel with the channel-closing conformation of Lys296 (cluster 2) hydrogen bonded to Ser186 and Glu181 and (d) in the channel-opening conformation hydrogen bonded to Tyr268 (cluster 3). Electrostatic surface potentials contoured at ±20kT/e, and negatively and positively charged surface areas in red and blue, respectively. Note that the positive charge of the ε-amino group of Lys296 (not shown) results in a positive surface potential of the neighbouring cavity (NC) in c, but is above the cut in d. Close-up view of Lys296 in (e) channel-closing (orange side chain – cluster 2) and (f) channel-opening conformation (light green side chain cut at the ε-amino group–cluster 3).</p

Constriction sites within the channel.

<p>Minimum inner width (d_min) measured at intervals of 0.6 Å progressing from opening A to opening B. The residues defining the constriction sites (C1–C4) are indicated, as well as the maximum and minimum extensions of the β-ionone moiety of the retinal.</p

Docking of retinal isomers to docking site II.

<p>Flexible docking of (a–c) 11<i>-cis-</i>retinal and (d–f) all-<i>trans</i>-retinal to site II, i.e. the retinal binding pocket (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g004" target="_blank">Figure 4</a>). The crystal structure of Ops* (PDB entry 3CAP) was used and full flexibility for Tyr191, Val204, Phe208, Phe273 and Lys296 side chains was allowed. The most likely conformation of (a) 11<i>-cis-</i>retinal and (d) all-<i>trans</i>-retinal is shown together with the neighbouring residues. The residues with altered conformation (orange) are super­imposed to the starting conformation. Cluster of docking poses of (b) 11<i>-cis-</i>retinal and (e) all-<i>trans</i>-retinal and (c, f) the respective list of ranked docking poses at different RMSD cut-off values (different colours identify individual poses). The best scored pose of the finally selected cluster (shaded) is shown with ball and sticks in a, b, d and e.</p

Docking of retinal isomers to docking site III.

<p>Flexible docking of (a–d) 11<i>-cis-</i>retinal and (e–h) all-<i>trans</i>-retinal to docking site III located close to opening B of the channel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004382#pone-0004382-g004" target="_blank">Figure 4</a>). The crystal structure of Ops* (PDB entry 3CAP) was used and full flexibility for Tyr191, Ile205, Phe208 and Phe273 side chains was allowed. The most likely conformation of (a, b) 11<i>-cis-</i>retinal and (e, f) all-<i>trans</i>-retinal shown with neighbouring residues from two different perspectives (TM5 and TM6 are depicted in cartoon representation). The residues with altered conformation (orange) are superimposed to the starting conformation. Cluster of docking poses of (c) 11<i>-cis-</i>retinal and (g) all-<i>trans</i>-retinal and (d, h) the respective list of ranked docking poses at different RMSD cut-off values (different colours identify individual poses). The best scored pose of the finally selected cluster (shaded) is shown with ball and sticks in a–c and e–g.</p

S1 Movie from It takes two transducins to activate the cGMP-phosphodiesterase 6 in retinal rods

Spatio-temporal development of PDE6 activation in the PBRD signal scenario