Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and ß-farnesene.

Mutational Analysis of (+)-Limonene Synthase.

The monoterpene limonene is produced by the enzyme limonene synthase in one of the simplest terpene cyclization reactions. The enzyme can use linalyl diphosphate (LPP) and neryl diphosphate (NPP) as substrates in addition to the naturally occurring substrate geranyl diphosphate (GPP), but the relationship among the three alternative substrates is not well understood. We explored the (+)-limonene synthase ((+)-LS) reaction using site-directed mutagenesis with the three different substrates (GPP, NPP, and LPP) to tease out details of the mechanism. In total, 23 amino acid positions in the active site of (+)-LS were targeted for mutation. In all cases, substitution with Ala resulted in a significant loss of enzyme activity using GPP or NPP as the substrate, but the mutations fell into two groups depending on the effect of using LPP as a substrate: group 1 mutations resulted in the loss of activity with all three substrates (GPP, NPP, and LPP); group 2 mutations resulted in loss of activity with GPP and NPP, but retained near-WT activity with LPP as a substrate. Importantly, mutations resulting in loss of activity with LPP but retention of activity with GPP and NPP were never observed. These data, in combination with the substrate order of reactivity for the WT enzyme (LPP > NPP > GPP), are consistent with a role for LPP as an intermediate in the (+)-LS reaction using either GPP or NPP as a substrate.

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase.

Most terpene synthase reactions follow Markovnikov rules for formation of high-energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 atom of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide the regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the nonreactive substrate analogue 12,13-difluorofarnesyl diphosphate, and determined the X-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is perfectly positioned to make a C-H···π interaction with the side chain benzene ring of residue F76; this would enhance hyperconjugation to stabilize a developing cation at C10 and thus support the anti-Markovnikov regioselectivity of the cyclization. The benzene ring is also positioned to catalyze the migration of H to C10 and stabilize a C9 carbocation. On the opposite face of C9, further cation stabilization is possible via interactions with the main chain carbonyl of I177 and the neighboring intramolecular C6═C7 bond. Mutagenesis experiments also support a role for residue 76 in these interactions, but most interesting is the F76W mutant, whose crystal structure clearly shows C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.

Opsins form a family of light-activated, retinal-dependent, G protein-coupled receptors (GPCRs) that serve a multitude of visual and nonvisual functions. Opsin 3 (OPN3 or encephalopsin), initially identified in the brain, remains one of the few members of the mammalian opsin family with unknown function and ambiguous light absorption properties. We recently discovered that OPN3 is highly expressed in human epidermal melanocytes (HEMs)-the skin cells that produce melanin. The melanin pigment is a critical defense against ultraviolet radiation (UVR), and its production is mediated by the Gαs-coupled melanocortin 1 receptor (MC1R). The physiological function and light sensitivity of OPN3 in melanocytes are yet to be determined. Here, we show that in HEMs, OPN3 acts as a negative regulator of melanin production by modulating the signaling of MC1R. OPN3 negatively regulates the cyclic adenosine monophosphate (cAMP) response evoked by MC1R via activation of the Gαi subunit of G proteins, thus decreasing cellular melanin levels. In addition to their functional relationship, OPN3 and MC1R colocalize at both the plasma membrane and in intracellular structures, and can form a physical complex. Remarkably, OPN3 can bind retinal, but does not mediate light-induced signaling in melanocytes. Our results identify a function for OPN3 in the regulation of the melanogenic pathway in epidermal melanocytes; we have revealed a light-independent function for the poorly characterized OPN3 and a pathway that greatly expands our understanding of melanocyte and skin physiology.

Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC.

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.

RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii.

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates.

The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. Although this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [ Devine et al. ( 2013 ) Proc. Natl. Acad. Sci. USA 110 , 13351 - 13355 ]. Because the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence-based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.

Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis.

Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.

Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands.

Recoverin (Rv), a small Ca(2+)-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca(2+) in both EF hands, but crystal structures have never been observed with Ca(2+) ions in both sites; all previous structures have Ca(2+) bound to only EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca(2+) ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and showed that two Ca(2+) ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca(2+) structure not seen previously and not seen in a second structure of the E153A mutant containing Na(+) instead of Ca(2+) in the EF2 site. The side-chain rearrangements in these residues form a 28 Å allosteric cascade along the surface of the protein connecting the Ca(2+)-binding site of EF2 with the active-site pocket responsible for binding RK.

Assembly of an activated rhodopsin-transducin complex in nanoscale lipid bilayers.

The formation and characterization of an activated complex of the visual pigment rhodopsin and its downstream signaling partner transducin have been the subject of intense focus by several research groups. While the subunit composition of the activated complex is still the subject of some controversy, our laboratory [Xie, G., D'Antona, A. M., Edwards, P. C., Fransen, M., Standfuss, J., Schertler, G. F. X., and Oprian, D. D. (2011) Biochemistry 50, 10399-10407] and that of Ernst et al. [Ernst, O. P., Gramse, V., Kolbe, M., Hofmann, K. P., and Heck, M. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 10859-10864] find that the two proteins are present in a 1/1 molar ratio. Unfortunately, these data could not distinguish a ratio of 1/1 from ratios of 2/2, 3/3, etc. For this reason, we reinvestigated the issue of stoichiometry of the activated complex, exploiting the ability of Nanodisc lipid bilayers to isolate single molecules of rhodopsin. We show here that the purified complex in Nanodiscs contains an activated rhodopsin with a covalently bound all-trans-retinal chromophore, that transducin has an empty nucleotide-binding pocket, that the isolated complex is active and dissociates upon addition of guanine nucleotide, and that the stoichiometry corresponds to exactly one molecule of rhodopsin and one molecule of transducin.

A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

Recoverin, a 23-kDa Ca(2+)-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca(2+)-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca(2+) binds preferentially to the R state; the myristoyl chain binds preferentially to the T state. In the absence of myristoylation, the R state predominates, and consequently, binding of Ca(2+) to the non-myristoylated protein is not cooperative. We show here that a mutation, C39A, of a highly conserved Cys residue among NCS proteins, increases the apparent cooperativity for binding of Ca(2+) to non-myristoylated recoverin. The binding data can be explained by an effect on the T/R equilibrium to favor the T state without affecting the intrinsic binding constants for the two Ca(2+) sites.

Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins.

Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution. To test for the existence of such a constraint, we asked whether it were possible to relocate the highly conserved Lys296 in the visual pigment bovine rhodopsin. We show here that the Lys can be moved to three other locations in the protein while maintaining the ability to form a pigment with 11-cis-retinal and activate the G protein transducin in a light-dependent manner. These results contradict the convergent hypothesis and support the homology of type I and type II rhodopsins by divergent evolution from a common ancestral protein.

Preparation of an activated rhodopsin/transducin complex using a constitutively active mutant of rhodopsin.

The interaction of rhodopsin and transducin has been the focus of study for more than 30 years, but only recently have efforts to purify an activated complex in detergent solution materialized. These efforts have used native rhodopsin isolated from bovine retina and employed either sucrose density gradient centrifugation or size exclusion chromatography to purify the complex. While there is general agreement on most properties of the activated complex, subunit stoichiometry is not yet settled, with rhodopsin/transducin molar ratios of both 2/1 and 1/1 reported. In this report, we introduce methods for preparation of the complex that include use of recombinant rhodopsin, so as to take advantage of mutations that confer constitutive activity and enhanced thermal stability on the protein, and immunoaffinity chromatography for purification of the complex. We show that chromatography on ConA-Sepharose can substitute for the immunoaffinity column and that bicelles can be used instead of detergent solution. We demonstrate the following: that rhodopsin has a covalently bound all-trans-retinal chromophore and therefore corresponds to the active metarhodopin II state; that transducin has an empty nucleotide-binding pocket; that the isolated complex is active and dissociates upon addition of guanine nucleotide; and finally that the stoichiometry corresponds reproducibly to a 1/1 molar ratio of rhodopsin to transducin.

The structural basis of agonist-induced activation in constitutively active rhodopsin.

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 Å for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal ß-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.

Crystal structure of a thermally stable rhodopsin mutant.

We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.

Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.

Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano. Lett. 2, 853-856). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry, which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein-coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10-nm-diameter lipid bilayer domain. Efficient transducin activation and isolation of a high affinity transducin-metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two-rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case, only one of the two receptors present in the Nanodisc was able to form a stable metarhodopsin II-G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.