Electron crystallography reveals the structure of metarhodopsin I

Rhodopsin is the prototypical G protein-coupled receptor, responsible for detection of dim light in vision. Upon absorption of a photon, rhodopsin undergoes structural changes, characterised by distinct photointermediates. Currently, only the ground-state structure has been described. We have determined a density map of a photostationary state highly enriched in metarhodopsin I, to a resolution of 5.5 A in the membrane plane, by electron crystallography. The map shows density for helix 8, the cytoplasmic loops, the extracellular plug, all tryptophan residues, an ordered cholesterol molecule and the beta-ionone ring. Comparison of this map with X-ray structures of the ground state reveals that metarhodopsin I formation does not involve large rigid-body movements of helices, but there is a rearrangement close to the bend of helix 6, at the level of the retinal chromophore. There is no gradual build-up of the large conformational change known to accompany metarhodopsin II formation. The protein remains in a conformation similar to that of the ground state until late in the photobleaching process.     

Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150mM KCl and 20 degrees C requires a surface potential change that corresponds to Deltasigma approximately -1.0e(-)/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.     

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state ²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific ²H labels have been introduced into the methyl groups of retinal and solid-state ²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent ²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state ²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.     

Changes in interhelical hydrogen bonding upon rhodopsin activation

Hydrogen bonding interactions between transmembrane helices stabilize the visual pigment rhodopsin in an inactive conformation in the dark. The crystal structure of rhodopsin has previously revealed that Glu122 and Trp126 on transmembrane helix H3 form a complex hydrogen bonding network with Tyr206 and His211 on H5, while the indole nitrogen of Trp265 on H6 forms a water-mediated hydrogen bond with Asn302 on H7. Here, we use solid-state magic angle spinning NMR spectroscopy to probe the changes in hydrogen bonding upon rhodopsin activation. The NMR chemical shifts of 15N-labeled tryptophan are consistent with the indole nitrogens of Trp126 and Trp265 becoming more weakly hydrogen bonded between rhodopsin and metarhodopsin II. The NMR chemical shifts of 15N-labeled histidine show that His211 is neutral; the unprotonated imidazole nitrogen is not coordinated to zinc in rhodopsin and becomes more strongly hydrogen bonded in metarhodopsin II. Moreover, measurements of rhodopsin containing 13C-labeled histidine show that a strong hydrogen bond between the side-chain of Glu122 and the backbone carbonyl of His211 is disrupted in metarhodopsin II. The implications of these observations for the activation mechanism of rhodopsin are discussed.     

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.     

Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.     

Equilibrium between metarhodopsin-I and metarhodopsin-II is dependent on the conformation of the third cytoplasmic loop

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln238 and Glu239, in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.     

Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from ¹³C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.     

Distribution of rhodopsin and retinochrome in the squid retina

The cephalopod retina contains two kinds of photopigments, rhodopsin and retinochrome. For many years retinochrome has been thought to be localized in the inner segments of the visual cells, whereas rhodopsin is in the outer segments. However, it is now clear that retinochrome can be extracted also from fragments of outer segments. In the dark-adapted retina of Loligo pealei retinochrome is distributed half-and-half in the inner and outer segments. Todarodes pacificus contains much more retinochrome than Loligo, and it is more abundant in the outer than in the inner segments. The outer segments of Loligo contain retinochrome and metarhodopsin in addition to rhodopsin, whether squids are kept in the dark or in the light. But there is extremely little metarhodopsin (about 3% of rhodopsin) even in light-adapted eyes. The inner segments contain only retinochrome, and much less in the light than in the dark. On the other hand, retinochrome in the outer segments increases markedly during light adaptation. These facts suggest the possibility that some retinochrome moves forward from the inner to the outer segments during light adaptation and there reacts with metarhodopsin to promote regeneration of rhodopsin.     

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.     

Photosensitivity spectrum of crayfish rhodopsin measured using fluorescence of metarhodopsin

Discrepancies exist among spectral measurements of sensitivity of crayfish photoreceptors, their absorption in situ, and the number and absorption spectra of crayfish photopigments that are extracted by digitonin solutions. We have determined the photosensitivity spectrum of crayfish rhodopsin in isolated rhabdoms using long wavelength fluorescence emission from crayfish metarhodopsin as an intrinsic probe. There is no measurable metarhodopsin in the dark-adapted receptor, so changes in the emission level are directly proportional to metarhodopsin concentration. We therefore used changes in metarhodopsin fluorescence to construct relaxation and saturation ("photoequilibrium") spectra, from which the photosensitivity spectrum of crayfish rhodopsin was calculated. This spectrum peaks at or approximately 530 nm and closely resembles the previously measured difference spectrum for total bleaches of dark-adapted rhabdoms. Measurements of the kinetics of changes in rhabdom fluorescence and in transmittance at 580 nm were compared with predictions derived from several model systems containing one or two photopigments. The comparison shows that only a single rhodopsin and its metarhodopsin are present in the main rhabdom of crayfish, and that other explanations must be sought for the multiple pigments seen in digitonin solution. The same analysis shows that there is no detectable formation of isorhodopsin in the rhabdom.     

Structural studies of metarhodopsin II,the activated form of the G-protein coupled receptor,rhodopsin

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.     

Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy

Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (G(t)). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G(t)/rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for G(t) from 64 nM to 0.7 nM, whereas G(t) affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of G(t) for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTP gamma S was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.     

Monomeric Rhodopsin Is the Minimal Functional Unit Required for Arrestin Binding

We have tested whether arrestin binding requires the G-protein-coupled receptor be a dimer or a multimer. To do this, we encapsulated single-rhodopsin molecules into nanoscale phospholipid particles (so-called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and β-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find that the monomeric rhodopsin in nanodiscs has a higher affinity for wild-type arrestin binding than does oligomeric rhodopsin in liposomes or nanodiscs, as assessed by stabilization of metarhodopsin II. Together, these results establish that rhodopsin self-association is not required to enable arrestin binding.     

Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications

The recently determined crystal structure of NhaA, the Na ⁺/H ⁺ antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.     

Early steps of the intramolecular signal transduction in rhodopsin explored by molecular dynamics simulations

We present molecular dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined X-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a negative charge. Restrained molecular dynamics simulations also provide evidence that the protein tightly confines the absolute conformation of the retinal around the C12-C13 bond to a positive helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, especially in helix VI. Our results suggest a possible molecular mechanism for the early steps of intramolecular signal transduction in a prototypical G-protein-coupled receptor.     

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the β-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant α-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular ³¹P-¹H and ¹H-¹H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.