From the
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany, the ¶International Institute of Molecular and Cell Biology and the Department of Chemistry, University of Warsaw, 1 Pasteur St, PL-02109 Warsaw, Poland, the ||Departments of Biological Structure, Biochemistry, and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195, and the **Departments of Ophthalmology, Pharmacology, and Chemistry, University of Washington, Seattle, Washington 98195
Received for publication, February 28, 2003 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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Initial deactivation of Meta II begins with the interaction of active rhodopsin with its receptor kinase, phosphorylation of the receptor, and a tight binding of arrestin to the still activated phosphorylated form of the receptor (6, 7). Full deactivation occurs when rhodopsin is regenerated. This requires the hydrolysis of the all-trans-retinylidene linkage and release of all-trans-retinal from the active site (1). Critical steps include the nucleophilic attack of water on the retinylidene bond within the hydrophobic binding site of rhodopsin and the diffusion of the hydrolyzed chromophore out of the binding pocket. Formation of opsin accompanies a significant increase in intrinsic Trp fluorescence after release of all-trans-retinal from the active site (8, 9). The retinal remains associated with opsin membranes and is converted by endogenous NADPH-dependent retinol dehydrogenase (RDH,1 reviewed in Ref. 1) to all-trans-retinol without further change in the intrinsic protein fluorescence (8). However, the stability of the complex between arrestin and phosphorylated Meta II may depend on the RDH activity (10, 11). In addition, during the Meta II decay a storage form of rhodopsin, metarhodopsin III (Meta III), is generated. Meta III and opsin are formed in parallel (8).
Studies on the effect of active site methylation (12) and palmitoylation of opsin on transducin (Gt) activation, and the retinoid competition on regeneration (13, 14), led to the proposal of a secondary binding site for all-trans-retinal within the opsin molecule. In this study, we demonstrate a site-directed retinal migration from the active site to an exit site during Meta II decay. Once this process has occurred, 11-cis-retinal is taken up into an entrance site and moved to the active site for rhodopsin regeneration. This ligand channeling mechanism is based firmly on the crystal structure of rhodopsin (15, 16), which allows identification of the putative secondary binding sites of the chromophore.
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EXPERIMENTAL PROCEDURES |
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Illumination ProtocolComplete bleaching (15 s at 495 nm) of rhodopsin membranes under conditions favoring Meta II was carried out at pH 6.0 (20 °C), and pH was adjusted to the desired value immediately after photoactivation (8). To reach a complete decay of Meta II, the sample was incubated for 60 min.
Fluorescence MeasurementsEmission spectra of intrinsic Trp fluorescence (SPEX Fluorolog 1680 instrument) were recorded between 310 and 400 nm using excitation at 295 nm and in BTP buffer, pH 6.5, at 20 °C. Samples were bovine serum albumin-stripped opsin membranes or solubilized opsin (0.1% DM) at a final concentration of 1 µM in 750 µl. In some samples, ethanolic solutions of retinoids (5 µl) were added in a stoichiometric amount to opsin. The excitation and emission slit bandpass were 0.4 and 7 nm, respectively. Low light intensities were used to prevent bleaching reactions of retinal or rhodopsin in the samples. For time-resolved records, fluorescence emission was monitored at 330 nm under the conditions outlined above. The fluorescence intensity was measured in counts per second (cps).
Protein and Chromophore ModelingA rhodopsin model suitable for simulations was built using the coordinates deposited in the Protein Data Bank under 1HZX [PDB] identifier (15). Hydrogen atoms were added in Insight II (v.2000, Accelrys, San Diego, CA). Missing loops of rhodopsin were created using Modeler module (20) of Insight II. Verifications of created loops and the whole structure were done using Profile-3D module (21) by calculating the compatibility between the sequence and the structure.
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RESULTS |
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11-cis-Retinal Uptake Precedes Schiff Base FormationThe interaction of retinal isomers with opsin was further analyzed by monitoring the time-dependent changes of intrinsic Trp fluorescence (Fig. 2). The complex formation between all-trans-retinal and membrane-bound opsin proceeded within seconds (t
5 s; Fig. 2, trace a). As expected (14), subsequent illumination of the opsin·all-trans-retinal complex did not result in any significant change in fluorescence.
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The uptake signal, as we term the rapid decrease in intrinsic protein fluorescence upon addition of retinoids, induced by addition of 11-cis-retinal to opsin membranes is biphasic (Fig. 2, trace b). The initial fast phase (t
6 s) is similar to the uptake signal seen with all-trans-retinal. The slower decrease in fluorescence intensity appeared to follow regeneration of rhodopsin (data not shown). This is confirmed by the fact that subsequent illumination led to an expected increase in fluorescence (Fig. 2, trace b) (8, 9), termed here the "release signal," that is specific only to retinoids that regenerate pigments such as 9-cis-retinal (trace d) (27, 28), and not 13-cis-retinal (trace c), or all-trans-retinal (trace a). The results demonstrate that both all-trans-retinal and 11-cis-retinal associate very rapidly with opsin to form opsin·retinal complexes. In the case of 11-cis-retinal, the complex is the precursor of the regenerated pigment.
In solubilized samples, the uptake signal with 11-cis-retinal is slower (t
140 s; Fig. 2, trace f). The signal likely correspond to regeneration of rhodopsin, in which the retinal bound in the original pocket, quenches Trp fluorescence. Consistently, a substantial fluorescence change was induced by illumination of the sample after completion of the uptake signal, showing that a considerable fraction of opsin was regenerated to light-sensitive rhodopsin under these conditions. Virtually no light-induced fluorescence changes are observed after incubation of solubilized opsin with all-trans-retinal or 13-cis-retinal (Fig. 2, traces e and g), while 9-cis-retinal (Fig. 2, trace h) had similar properties to those of 11-cis-retinal.
Uptake of Retinal After Decay of Meta IIThe decay of Meta II is accompanied by a significant increase in fluorescence, both in detergent (8, 9) and in native membranes (8). This is the release signal, reflecting the release of all-trans-retinal from the active site. Here, Meta II formed in membranes was allowed to decay for 60 min without removal of the endogenous photoisomerized chromophore. Subsequent addition of exogenous all-trans-retinal at 1:1 ratio to opsin (Fig. 3A) produced a normal uptake signal, although an amount equimolar to opsin was already present in the membranes. The signal had the same amplitude as was measured with purified opsin without endogenous retinal (Fig. 2, trace a). A second illumination after addition of exogenous all-trans-retinal and incubation had no effect on opsin fluorescence. However, when the membranes were solublized with DM, a biphasic increase in fluorescence intensity was observed (Fig. 3A). The amplitude of the fast initial jump is comparable with the amplitude of the uptake signal, suggesting that the detergent causes dissociation of the complex between opsin and the exogenously added all-trans-retinal. Subsequently, the fluorescence increased to a high Trp emission intensity, which is typical for solubilized opsin (see Fig. 1). Consistently, the release signal from a solubilized light-activated rhodopsin sample was larger (data not shown).
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A large, biphasic uptake signal was observed when 11-cis-retinal was used instead of all-trans-retinal (Fig. 3B). These results are similar to the observation made for opsin depleted of endogenous chromophore (see Fig. 2, trace b). A second illumination evoked a small release signal. The addition of detergent increased the fluorescence to the same maximum level as was seen with the all-trans isomer. No significant uptake signals were observed upon addition of retinoids to dark-adapted rhodopsin membranes (data not shown).
The observation of a normal uptake signal after Meta II decay demonstrates that there is a rapid complex formation between the exogenously added all-trans-retinal/11-cis-retinal and the decay product of Meta II. The retinal released in the course of Meta II decay does not diffuse freely. If this were the case, it would necessarily equilibrate, thus reducing or even canceling the uptake signal. Even if we assume that part of the released chromophore bound to binding sites other than on opsin, the same would apply to the exogenously provided chromophore. Consistently, all measured uptake signals (including that with empty apoprotein and with the Meta II decay product) had the same amplitude. Moreover, the absolute level of fluorescence intensity of purified empty opsin was similar to the Meta II decay product (i.e. opsin containing endogenous all-trans-retinal). These data argue for two independent binding sites for retinals on the opsin molecule (entrance and exit sites, see "Discussion"), in addition to the original binding pocket bearing the 11-cis-retinylidene/all-trans-retinylidene linkage.
A similar protocol was employed with all-trans-retinol. The uptake signal was again observed both with decayed Meta II and isolated opsin apoprotein (Fig. 3C), demonstrating that all-trans-retinol quenches opsin fluorescence like the retinal isomers. The release signal of all-trans-retinal is also seen in the presence of NADPH (Fig. 3D). The amplitude of the signal is reduced due to the quenching effect of NADPH but is similar when normalized to initial fluorescence intensity (see Ref. 8). This result suggests that all-trans-retinol enzymatically formed during Meta II decay, like endogenous all-trans-retinal, may not be entirely free to diffuse.
Crystal Structure of Rhodopsin Offers Potential Binding Sites for RetinoidsIn the crystal structure of rhodopsin, two heptane-1,2,3-triol (HTO) molecules were identified that bind to two hydrophobic cavities (Fig. 4a). One of them is located in a large cavity, which also easily accommodates 11-cis-retinal (311 Å3) and all-trans-retinal (309 Å3), close to the palmitoylation site within the transmembrane segment (termed site II). For the all-trans-retinal, the contact residues include Met317, Val318, and Leu321 of helix 8 around the polyene chain and Leu50, Ile54 (helix 1), Val304, and Met308 (helix 7) around the -ionone ring (Fig. 4b). Cys322 and Leu328 of the C-terminal loop are close to the oxygen atom of retinal. Site II for the cis-retinoid consists of Phe52, Pro53, Phe56, Leu57, Gln64, and Tyr60 of helix 1. Thr 320 and Lys339 of the C-terminal loop are close to the oxygen atom of retinal. Leu321 divides the cis-from the trans-site and is covered by palmitoyl chains. It is plausible that these sites are occupied by detergent when membranes are solubilized, competing out the retinoid effects as demonstrated in the study. The second site is located on the cytoplasmic surface of rhodopsin (site III) (Fig. 4c). It encompasses Asn315, Cys316, Thr319 (helix 8), Lys325, Asn326, Leu328, and Gly329 (C-terminal loop) and residues around the
-ionone include Ala333, Thr336, Val337, Ser338, Glu341, and Thr342. The size of site II is large enough to accommodate all-trans-retinal and 11-cis-retinal individually or together (Fig. 4b), while the smaller site III can only bind one all-trans-retinal molecule (Fig. 4c). Site III is different from the HTO-binding site located in the main cavity on the rhodopsin cytoplasmic surface, which is more hydrophilic than site III. In agreement with the modeling, all-trans-retinal is likely to bind to the more hydrophobic cavity termed site III.
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DISCUSSION |
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During Decay of Meta II, all-trans-Retinal Is Released from the Active Site to an Exit SiteA release of the chromophore from the original binding site (site I) can be observed by an accompanying change in the intrinsic Trp fluorescence of the apoprotein (this study and Refs. 8 and 9). According to current understanding, all-trans-retinal is released to the cytoplasm and then reduced to all-trans-retinol by a membrane-bound RDH (1). A fraction of all-trans-retinal may escape into the intradiscal space, where it is removed to the cytoplasm by an ABCR transporter protein (reviewed in Refs. 1 and 29).
A first surprising result of the current analysis is that opsin, embedded in the lipid host of the membrane, does not release its photoisomerized chromophore into the bulk lipid phase. Instead, opsin keeps the chromophore bound in a site termed the "exit" site, preventing all-trans-retinal to diffuse freely. Endogenous all-trans-retinal does not migrate to the entrance site and does not cancel the release signal, while exogenously added retinoids bind to the entrance site, suppressing Trp fluorescence. Moreover, the persistent occupation of the exit site by endogenous all-trans-retinal is not merely due to a higher affinity of the exit site compared with the entrance site. If this were the case, exogenously added retinoid (1:1 ratio of retinoid to opsin) would preferentially bind to the exit site, thus not causing a quenching of fluorescence. The data in Fig. 3 show that after completion of the release signal, the binding signal is the same as with opsin, i.e. with empty active and release sites (Fig. 2). We conclude that the exit site remains closed for added retinoids, and then filling of the entrance site does not require emptying of the exit site.
Reduction of all-trans-Retinal Can Occur in the Exit Site Although all-trans-retinol evokes an uptake signal when exogenously added, release signal of all-trans-retinal in the presence of NADPH and subsequent uptake signal of exogenous retinol were again of the same size (Fig. 3, C and D). This indicates that during Meta II decay and subsequent enzymatic reduction, neither all-trans-retinal nor -retinol became free (see above). We may infer that, at least under the experimental conditions, even all-trans-retinol remained bound to the exit site. This would in turn allow the conclusion that reduction involves opsin·RDH complex formation. Although we have not yet attempted to study the complex with arrestin, reduction of the opsin-bound photoisomerized chromophore would open the possibility that both arrestin and RDH bind. This observation would explain why arrestin does not inhibit the visual cycle in vivo (30).
Retinoids Are Taken Up into an Entrance SiteThe release of all-trans-retinal and also the uptake of retinoids accompany a change in intrinsic protein fluorescence. The quench effect upon uptake is observed in native disc membranes, but not in the presence of detergent (Fig. 1). The uptake signal is observed with both all-trans-retinal and 11-cis-retinal when they are added to opsin devoid of any endogenous retinoid (Figs. 1 and 2) or after the decay of Meta II (Fig. 3). For a 1:1 ratio of retinal to opsin, the signal is of approximately the same size as the release signal. An obvious explanation would be that the added retinal re-fills the active site during Schiff base formation. However, this cannot be the case because the fluorescence change is 20 times faster than rhodopsin regeneration. The kinetics indicate a separate complex formation step, different from the formation of ground state rhodopsin. This would still leave open the possibility that the retinoids enter the active site and form a non-covalent association inside the pocket (site I). In such a model, an assumption would be that in a subsequent slower reaction, 11- or 9-cis-retinal would form the Schiff base from this pre-bound state. However, excess of all-trans-retinal or all-trans-retinol do not inhibit formation of rhodopsin (14, 31, 32). This observation excludes any significant occupation of the active site by all-trans-retinal during uptake into opsin and proves that, for all-trans-retinal, the uptake signal cannot be interpreted as an uptake into the active site. Consistent with these observations, all-trans-retinal and 13-cis-retinal, which do not regenerate rhodopsin because they do not fit the active site of opsin (33), also evoke a detergent-sensitive uptake signal (Fig. 2). Because the uptake yielded a signal with the same amplitude and similarly fast kinetics, not only with all-trans-but also with 11-cis- and 9-cis-retinal, we infer that 11-cis-retinal also passes through an "entrance" site(s) before it migrates to the active site and forms the Schiff base. In the case of 11-cis-retinal, it is channeled to the active site, causing a transient activation of opsin (34) before the 11-cis-reinylidene linkage is formed. This reaction could be aided by two residues, Glu181 and Glu113, which may act as general bases.
Ligand Channeling in OpsinThe scheme in Fig. 5A interprets the findings based on the notion that retinals are unidirectionally passed through the three sites identified above (ligand-channeling hypothesis). The reaction pathway starts with rhodopsin (activation state), in which 11-cis-retinal is covalently bound via a protonated Schiff base to the active site and the intrinsic Trp fluorescence is fully quenched. Light absorption leads through retinal isomerization to Meta II (active state). Despite the activating conversion, the net change in fluorescence between rhodopsin and Meta II is very small (8). A large change in Trp fluorescence (relief from fluorescence quenching) occurs in the subsequent Meta II decay. In parallel to Meta III (storage state) formation, exit and entrance sites are now set to function. Channeling all-trans-retinal into the exit site and opening the entrance for various retinoids generates a native form of opsin, the opsin (exit) state. In this state, the RDH enzyme has access to its all-trans-retinal substrate and can form all-trans-retinol with NADPH as a cofactor. The cycle is closed when fresh 11-cis-retinal binds to the entrance site, to form the opsin·11-cis-retinal complex (entrance state), which enables regeneration of rhodopsin. The driving force for retinal channeling is provided by the light reaction, which is also the link between the two ducts for cis- and trans-retinal, respectively (Fig. 5A), and secures the unidirectionality of the overall process.
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When fresh 11-cis-retinal binds to the entrance, the exit site can release all-trans-retinal or all-trans-retinol. In this condition, the all-trans species may also bind to the entrance site (of another opsin molecule) (14). The complex formed in vitro (Fig. 5B), for which an activity toward the G-protein (12, 14, 25) and rhodopsin kinase (35) has been demonstrated, may be identified with the state with all-trans-retinal in the entrance site. It is important to note that all-trans-retinal bound to the entrance site does not inhibit the regeneration of rhodopsin (14). In agreement with the modeling (see below), this implies a more complex structure of the entrance domain, with a site to which all-trans-retinal can bind without inhibition of the duct for 11-cis-retinal permeation. A consequence of the above reaction scheme is that empty opsin is unlikely to exist in situ. Only harsh treatment in vitro, e.g. by washing with bovine serum albumin or treatment with hydroxylamine, can fully strip the exit site. Finally, it must be recognized that all-trans-retinal added in excess forms both covalent and non-covalent complexes with opsin as documented in previous studies (11, 12).
Assignment of Ligand Channeling to Rhodopsin Structure The ligand channeling found in this study is reminiscent of the tunneling of substrate found in some hydrophobic ligand-binding proteins, such as phosphatidyl-transfer protein (36). Most likely, the selectivity comes about through a specific opsin conformation that arises when all-trans-retinal is released from the active site. This conformational change is a late consequence of photon absorption and cannot be induced once opsin is stripped of endogenous all-trans-retinal and collapsed to a new conformation. Such a transformation would allow unidirectional uptake and release of the chromophore. In a previous investigation of the opsin·all-trans-retinal complex (14), we had found that all-trans-retinal-induced opsin activity toward the G-protein depended on palmitoylation of Cys322 and Cys323. When combining this with the present results, one would identify site II as the entrance, and would suggest site III as the exit. In native opsin, only 11-cis-retinal can pass the tunnel to the active site I. Only for certain mutants, including E113Q (37), the all-trans isomer has access to site I.
According to modeling, either all-trans- or 11-cis-retinal can bind to site II in rhodopsin, but it would take opsin to allow all-trans- or 11-cis-retinal to induce the conformational change seen in the fluorescence uptake signal. The detailed mechanism by which the conformational change translates into a fluorescence change remains to be elucidated. There is a large distance between the entrance site and Trp265, which resides in site I and is the interaction partner of the -ionone in the dark state. Therefore, we must envisage an indirect mechanism for the quenching related to retinal uptake. There is an additional change in fluorescence when 11-cis-retinal passes from the entrance to the active site. 11-cis-retinal may reorient itself during Schiff base formation, thus tightly immobilizing Trp265 (38).
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CONCLUSIONS |
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FOOTNOTES |
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Both authors contributed equally to this work.
A RPB Senior Investigator and a recipient of the Humboldt Research Award for Senior United States Scientists.
To whom correspondence should be addressed: Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany. Tel.: 49-30-450-524111; Fax: 49-30-450-524952; E-mail: kph{at}charite.de.
1 The abbreviations used are: RDH, retinol dehydrogenase; BTP, bis-Tris-propane; Gt, photoreceptor G-protein, transducin; HTO, heptane-1,2,3-triol; Meta II, metarhodopsin II (photoactivated rhodopsin); Meta III, metarhodopsin III; DM, n-dodecyl--maltoside; Pipes, 1,4-piperazinediethanesulfonic acid; ROS, rod outer segment.
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ACKNOWLEDGMENTS |
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REFERENCES |
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