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

Uptake and release of the retinal ligand into the retinal photoreceptor opsin

G-Protein-gekoppelte Rezeptoren (GPCRs) sind die größte Gruppe der Zelloberflächenrezeptoren in Vertebraten. Rhodopsin ist der am besten untersuchte GPCR und diente lange als Vorlage für das Verständnis anderer GPCRs. Rhodopsin besteht aus dem Apoprotein Opsin und dem inversen Agonisten 11-cis-Retinal, der über eine Schiff’sche Base kovalente mit der Lys296-Seitenkette des Opsins verknüpft ist. Die lichtinduzierte Isomerisierung des 11-cis-Retinals zum Agonisten all-trans-Retinal führt zur Aktivierung des Rhodopsins. Nach der Entstehung der aktiven Konformation (Meta II) wird die Schiff’sche Base zwischen all-trans-Retinal und Opsin hydrolysiert und all-trans-Retinal verlässt das Protein. Anschließend kann 11-cis-Retinal aufgenommen werden, wodurch sich wiederum Rhodopsin bildet. Um die zugrunde liegenden Mechanismen der Rhodopsinregeneration und des Meta II-Zerfalls besser zu verstehen, wurde in dieser Arbeit die Interaktion von Opsin mit seinen Retinalliganden untersucht. Ein Teil dieser Arbeit beschreibt die Aufnahme und Abgabe des Retinals durch Opsin. Die Kristallstrukturen des Meta II und aktiven Opsins ließen zwei Öffnungen eines mutmaßlichen Ligandenkanals erkennen, der die Retinalbindungstasche mit der hydrophoben Membranumgebung verbindet. Die Existenz dieses Kanals und die Rolle der zwei Öffnungen bei der Aufnahme und Abgabe des Retinals wurden durch die ortsgerichtete Mutagenese experimentell überprüft. Die Ergebnisse zusammengenommen zeigten, dass die Mutationen nicht lokal auf die Kanaldurchlässigkeit wirken, sondern weitreichende Effekte auf die Struktur des gesamten Rezeptors haben. In dieser Arbeit wurde ein Modell für die Rezeptor-Retinal-Interaktion entwickelt, in dem die aktive Rezeptorkonformation (Opsin*) für das Öffnen des Retinalkanals notwendig ist. Darüber hinaus kann gesagt werden, dass der geschwindigkeitsbestimmende Schritt bei der Bildung des Rhodopsins oder beim Zerfall des Meta II die richtige Positionierung des Liganden innerhalb der Bindungstasche am Lys296 ist. Ein weiterer Teil dieser Arbeit beschreibt die Rolle des Opsin* bei der Retinalaufnahme. Die Bindung des C-terminalen Endes des Gtα-Proteins führt zur Verschiebung des Opsin/Opsin*-Gleichgewichts und stabilisiert Opsin*. Die Untersuchungen zeigten, dass die Opsin*-Konformation all-trans-Retinal reversibel aufnehmen und es über eine Schiff’sche Base kovalent an Lys296 binden kann. Basierend auf diesen Untersuchungen kann Opsin auch als ein Rezeptor für diffusionsfähige Liganden aufgefasst werden. Ein letzter Teil dieser Arbeit beschreibt die Untersuchung der Hydrolyse der Schiff’schen Base durch die Verwendung von Hydroxylamin (HA) und seinen Derivaten. Die Ergebnisse zeigten, dass HA den Zerfall der lichtaktivierten Metarhodopsinspezies beschleunigte, während größere Derivate keinen signifikanten Einfluss hatten. Diese Beobachtungen lassen vermuten, dass ein Wasserkanal existiert, der sich nach der Lichtaktivierung des Rhodopsins öffnet und extrem stringent in Bezug auf die Größe und Polarität der einströmenden Substanzen ist. Zusammenfassend demonstrieren diese Ergebnisse, dass die aktive Konformation des Rezeptors eine zentrale Rolle bei der Aufnahme und Abgabe des Retinalliganden spielt. Während die Hydrolyse und Abgabe des all-trans-Retinals durch die aktive Rezeptorkonformation induziert wird, folgt die Aufnahme des 11-cis und all-trans-Retinals verschiedenen Mechanismen. Diese Arbeit gibt Aufschluss über die komplexen kinetischen und strukturellen Mechanismen, die den Aufnahme- und Abgabeprozess des Retinalliganden steuern.G protein-coupled receptors (GPCRs) are one of the largest groups of cell surface receptors in vertebrates. Rhodopsin is the best investigated GPCR and has long stood as a model for understanding other GPCRs. Rhodopsin consists of the apoprotein opsin and the inverse agonist 11-cis-retinal, which is Schiff base-linked to residue Lys296. Light-induced isomerisation of 11-cis-retinal to the agonist all-trans-retinal leads to activation of rhodopsin. After formation of the active conformation (Meta II), the retinylidene Schiff base is hydrolysed and all-trans-retinal is released from opsin. 11-cis-retinal is then taken up, which reforms rhodopsin. In this thesis the interaction of opsin with its retinal ligands was investigated to better understand the underlying mechanisms of rhodopsin regeneration and Meta II decay. A first part of this thesis explores the uptake and release of retinal from opsin. The crystal structures of Meta II and active opsin revealed two openings of a presumable ligand channel which connects the retinal binding pocket with the hydrophobic membrane environment. The existence of this ligand channel and the role of the openings in the uptake and release of retinal were experimentally tested by site-directed mutagenesis. Overall the results showed that the mutations did not have local effects on channel permeability, but rather had long ranging effects on the entire receptor structure. This study developed a model of receptor-ligand interactions, in which the active receptor conformation (Opsin*) is necessary for the retinal channel to be open. Furthermore, the rate limiting step for either the formation of rhodopsin or Meta II decay is the correct positioning of ligand within the binding pocket relative to Lys296. In a second presented part of this thesis, the role of Opsin* in retinal uptake was further explored. Binding of the c-terminal end of Gtα protein was found to stabilize Opsin* relative to inactive opsin. Opsin* was observed to take up all-trans-retinal and form a retinylidene Schiff base with Lys296. Based on these observations, opsin can be understood as a receptor for diffusible ligands. In a final presented part of this thesis, retinal Schiff base hydrolysis was investigated using hydroxylamine (HA) and its alkylated derivatives. Briefly, HA accelerated the decay of light-activated metarhodopsin species, while the larger derivatives had little influence. The results imply the existence of a water channel,which is opened in light-activated rhodopsin and is extremely stringent with regard to size and polarity of inflowing substances. In summary, these results demonstrate that the active conformation of the receptor plays a central role in the uptake and release of retinal. While the hydrolysis and release of all-trans-retinal is induced by the active receptor conformation, the uptake of 11-cis- and all-trans-retinal follows different mechanisms. The work presented in this thesis sheds light on the complex kinetic and structural mechanisms governing the uptake and release process of the retinal ligand

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

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

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

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

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