13 research outputs found
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<sup>GDP</sup> with an unstructured α5 C-terminus encounters R* with a partially unstructured cytoplasmic crevice. (B) The intermediate R*•G<sup>GDP</sup> 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<sup>GDP</sup>. (C) Rotation of α5 lowers the energy barrier separating R*•G<sup>GDP</sup> from nucleotide free R*•G<sup>empty</sup> resulting in GDP release. (D) Uptake of GTP and dissociation of G<sup>GTP</sup> completes the nucleotide exchange reaction.</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 β<sub>2</sub>AR* 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<sup>3.50</sup> (orange patch) located at the floor of the cytoplasmic crevice. TM helices are shown as cylinders. For clarity, H8 and H6 of β<sub>2</sub>AR* 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<sup>3.50</sup> or <b>(F)</b> between the carbonyl oxygen of C347 of GtαCT and R135<sup>3.50</sup>. Gray bars indicate the range of mobility of GαCT in MD simulations of the X-ray structures of (left) holo β<sub>2</sub>AR*•Gs<sup>empty</sup> (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 β<sub>2</sub>AR*•Gs<sup>empty</sup> (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
Comparison of the β<sub>2</sub>AR*•Gs<sup>GDP</sup> model (left panel) and the β<sub>2</sub>AR*•Gs<sup>empty</sup> 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 β<sub>2</sub>AR* are colored in black, from GsαCT in red. Potential hydrogen bonds are denoted as black dashed lines. <b>(E)</b> Complete model of the β<sub>2</sub>AR*•Gs<sup>GDP</sup> intermediate compared to <b>(F)</b> the β<sub>2</sub>AR*•Gs<sup>empty</sup> X-ray structure (PDB entry 3SN6). R*•G<sup>GDP</sup> was obtained by superposition of Gsα<sup>GTPγS</sup> (PDB entry 1AZT) with the intermediate β<sub>2</sub>AR*•GsαCT complex by common backbone atoms. Black arrows indicate the rotation of α5.</p
Data_Sheet_1_Binding, Thermodynamics, and Selectivity of a Non-peptide Antagonist to the Melanocortin-4 Receptor.doc
<p>The melanocortin-4 receptor (MC4R) is a potential drug target for treatment of obesity, anxiety, depression, and sexual dysfunction. Crystal structures for MC4R are not yet available, which has hindered successful structure-based drug design. Using microsecond-scale molecular-dynamics simulations, we have investigated selective binding of the non-peptide antagonist MCL0129 to a homology model of human MC4R (hMC4R). This approach revealed that, at the end of a multi-step binding process, MCL0129 spontaneously adopts a binding mode in which it blocks the agonistic-binding site. This binding mode was confirmed in subsequent metadynamics simulations, which gave an affinity for human hMC4R that matches the experimentally determined value. Extending our simulations of MCL0129 binding to hMC1R and hMC3R, we find that receptor subtype selectivity for hMC4R depends on few amino acids located in various structural elements of the receptor. These insights may support rational drug design targeting the melanocortin systems.</p
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 EÂ(D)ÂRY and Yx<sub>7</sub>KÂ(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
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
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
Constriction sites within the channel.
<p>Minimum inner width (d<sub>min</sub>) 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