Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes

G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65-/- mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.     

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.     

Crystallization of isoelectrically homogeneous cholera toxin

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.     

Rhodopsin and phototransduction: a model system for G protein-linked receptors.

Rhodopsin is the photoreceptor protein in rod cells of the vertebrate retina and the first member of the class of G protein-coupled receptors for which the amino acid sequence was determined. Rhodopsin is available in greater quantities than any other receptor of its class and therefore has been studied biochemically and biophysically by methods difficult or impossible to apply to its fellow receptors. Such studies support a model in which rhodopsin consists of seven transmembrane helices that form a binding pocket for its ligand, 11-cis retinal. Insights into the structure and function of rhodopsin serve as a model for understanding the structure and function of other members of the receptor class. Rhodopsin undergoes a change in conformation upon photoexcitation and activates a G protein, transducin, and is phosphorylated by a receptor-specific kinase, rhodopsin kinase. The phosphorylated photoactivated rhodopsin is bound by arrestin, thereby terminating activity of the receptor in the signal transduction process. These auxiliary proteins that function with rhodopsin on rod cells serve as models for understanding how other members of the receptor family may function in conjunction with other G proteins, kinases, and arrestin-like proteins.     

Production of (+)-globulol needle crystals on the surface mycelium of Quambalaria cyanescens

The structure of unique colorless needle crystals growing from the surface mycelium of the basidiomycete Quambalaria cyanescens and identified as (+)-globulol was followed by mass spectrometry, X-ray diffraction, and polarimetry. The mechanism of (+)-globulol fragmentation is proposed based on collision induced dissociation mass spectrometry. X-Ray analysis revealed that crystal packing is governed by hydrogen bond O-H.....O connecting the molecules into an infinite helix along a 3-fold screw axis propagating along the longest dimension of the needle crystal (c-axis of the unit cell). The X-ray diffraction data correspond well with the proposed structure determined by mass spectrometry.     

The molecular and crystal structure of dextrans: a combined electron and X-ray diffraction study. II. A low temperature, hydrated polymorph

The crystal and molecular structure of a dextran hydrate has been determined through combined electron and X-ray diffraction analysis, aided by stereochemical model refinement. A total of 65 hk0 electron diffraction intensities were measured on frozen single crystals held at the temperature of liquid nitrogen, to a resolution limit of 1.6 A. The X-ray intensities were measured from powder patterns recorded from collections of the single crystals. The structure crystallizes in a monoclinic unit cell with parameters a = 25.71 A, b = 10.21 A, c (chain axis) = 7.76 A and beta = 91.3 degrees. The space group is P2(1) with b axis unique. The unit cell contains six chains and eight water molecules, with three chains of the same polarity and four water molecules constituting the asymmetric unit. Along the chain direction the asymmetric unit is a dimer residue; however, the individual glucopyranose residues are very nearly related by a molecular 2-fold screw axis. The conformation of the chain is very similar to that in the anhydrous structure, but the chain packing differs in the two structures in that the rotational positions of the chains about the helix axes (the chain setting angles) are considerably different. The chains still pack in the form of sheets that are separated by water molecules. The difference in the chain setting angles between the anhydrous and hydrate structures corresponds to the angle between like unit cell axes observed in the diffraction diagrams recorded from hybrid crystals containing both polymorphs. Despite some beam damage effects, the structure was determined to a satisfactory degree of agreement, with the residuals R'(electron diffraction) = 0.258 and R(X-ray) = 0.127.     

Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase

Periplasmic Escherichia coli L-asparaginase II with Y25F mutation in the active-site cavity has been obtained by recombinant techniques. The protein was crystallized in a new hexagonal form (P6(5)22). Single crystals of this polymorph, suitable for X-ray diffraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipitant (pH 4.8). The crystals are characterized by a = 81.0, c = 341.1 A and diffract to 2.45 A resolution. The asymmetric unit contains two protein molecules arranged into an AB dimer. The physiologically relevant ABA'B' homotetramer is generated by the action of the crystallographic 2-fold axis along [1, -1, 0]. Kinetic studies show that the loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs kcat without significantly affecting Km.     

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.     

Crystallization and preliminary X-ray analysis of porcine synovial collagenase

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Crystallization of a scRIP—gelonin isolated from plant seeds Gelonium multiforum

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P2₁, with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along ψ =13° and ? =88°. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein. © 1994 Wiley-Liss, Inc.     

Characterization and crystal packing of three-dimensional bacteriorhodopsin crystals

The three-dimensional crystals of the integral membrane protein bacteriorhodopsin have been characterized by X-ray diffraction and freeze-fracture electron microscopy: the needle-like form A crystals belong to space group P 1 (pseudohexagonal) with seven molecules per crystallographic unit cell forming one turn of a non-crystallographic helix. The probable arrangement of the bacteriorhodopsin molecules is derived from freeze-fracture electron micrographs and chromophore orientation. Membrane-like structures are not present. The same helices of bacteriorhodopsin molecules found in crystal form A also make up the cube-like crystal form B. They are now arranged in all three mutually perpendicular directions. These cubes are always highly disordered, since the unit cell length corresponds to 6.7 molecules of the 7-fold helix. Very often, conversion of bacteriorhodopsin from the three-dimensional crystals into filamentous material occurs.     

Structure of tropomyosin at 9 angstroms resolution.

We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.     

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.     

Crystallization and preliminary X-ray analysis of complexes of peptide inhibitors with human recombinant and mouse submandibular renins.

Inhibitor-complexed crystals of mouse and human renins suitable for X-ray analysis have been prepared. The mouse renin is complexed with a non-hydrolysable decapeptide analogue of rat angiotensinogen containing a hydroxyethylene isostere in place of the scissile bond. The crystals are monoclinic, space group P2(1) with cell dimensions a = 78.3 A, b = 117.8 A, c = 85.9 A, beta = 101.18 degrees containing four molecules per asymmetric unit. The human renin is fully glycosylated and complexed with a tetrapeptide containing norstatine. The complex crystallises in the cubic space group P2(1)3 with a = 143.1 A and has two molecules in the asymmetric unit. The rotation function of the mouse renin complex indicates pseudo 222 symmetry while that of human renin indicates a pseudo 2-fold axis. Full structural analyses of the two complexes are underway.     

Crystal Structure of Escherichia coli-Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form

Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein''s fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.     

Packing analysis of crystalline myosin subfragment-1. Implications for the size and shape of the myosin head

Crystals of myosin subfragment-1 have been examined by X-ray diffraction and electron microscopy to determine how the molecules pack in the unit cell and to gain preliminary information on the size and shape of the myosin head. Subfragment-1 crystallizes in space group P212121. Analysis of the X-ray diffraction photographs shows that there are eight molecules in the unit cell with two in the asymmetric unit related by a non-crystallographic or local 2-fold axis. It also indicates that in projection down the a axis, two molecules of myosin subfragment-1 lie almost directly on top of one another except for a translation of about 9 A along c. Small crystals were fixed and embedded in the presence of tannic acid, and thin sections were cut perpendicular to each of the three crystallographic axes. Image analysis of micrographs recorded from these sections confirm the packing arrangement deduced from X-ray diffraction, and give the approximate size and shape of the molecule in the crystal lattice. They show that the molecule is at least 160 A long with a maximum thickness of about 60 A, and that it has marked curvature in the unit cell.