The 8.5A projection structure of the core RC-LH1-PufX dimer of Rhodobacter sphaeroides

Two-dimensional crystals of dimeric photosynthetic reaction centre-LH1-PufX complexes have been analysed by cryoelectron microscopy. The 8.5A resolution projection map extends previous analyses of complexes within native membranes to reveal the alpha-helical structure of two reaction centres and 28 LH1 alphabeta subunits within the dimer. For the first time, we have achieved sufficient resolution to suggest a possible location for the PufX transmembrane helix, the orientation of the RC and the arrangement of helices within the surrounding LH1 complex. Whereas low-resolution projections have shown an apparent break in the LH1, our current map reveals a diffuse density within this region, possibly reflecting high mobility. Within this region the separation between beta14 of one monomer and beta2 of the other monomer is approximately 6A larger than the average beta-beta spacing within LH1; we propose that this is sufficient for exchange of quinol at the RC Q(B) site. We have determined the position and orientation of the RC within the dimer, which places its Q(B) site adjacent to the putative PufX, with access to the point in LH1 that appears most easily breached. PufX appears to occupy a strategic position between the mobile alphabeta14 subunit and the Q(B) site, suggesting how the structure, possibly coupled with a flexible ring, plays a role in optimizing quinone exchange during photosynthesis.     

A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

Dimers of light-harvesting complex 2 from Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy

Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein complexes to be arranged in a densely packed energy-transducing network. Protein packing may play a determining role in the formation of functional photosynthetic domains and membrane curvature. To further investigate in detail the packing effects of like-protein photosynthetic complexes, we report an atomic force microscopy investigation on artificially created 2D crystals of the peripheral photosynthetic light-harvesting complexes 2 (LH2's) from the bacterium Rhodobacter sphaeroides. Instead of the usually observed one or two different crystallization lattices for one specific preparation protocol, we find seven different packing lattices. The most abundant crystal types all show a tilting of LH2. Most surprisingly, although LH2 is a monomeric protein complex in vivo, we find an LH2 dimer packing motif. We further characterize two different dimer configurations: in type 1, the LH2's are tilted inwards, and in type 2, they are tilted outwards. Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2's that constitute a lattice of zig-zagging LH2's. In addition, analyses of the tilt of the LH2's within the zig-zag lattice and that observed within the dimers corroborate their similar packing motifs. The type 2 dimer configuration exhibits a tilt that, in the absence of up-down packing, could bend the lipid bilayer, leading to the strong curvature of the LH2 domains as observed in Rhodobacter sphaeroides photosynthetic membranes in vivo.     

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.     

Carotenoid stoichiometry in the LH2 crystal: no spectral evidence for the presence of the second molecule in the alpha/beta-apoprotein dimer

In this work we have investigated the carotenoid-protein interactions in LH2 complexes of Rhodopseudomonas acidophila both in "free in solution" mixed-micelles and in three-dimensional crystals by Raman spectroscopy in resonance with the carotenoid (Car) molecules. We show that the Car molecules when bound to their binding pockets show no significant differences when the complexes are "free in solution" or packed in crystalline arrays. Furthermore, there is no significant wavelength dependence in the Raman spectrum of the Car molecules of LH2. This indicates that there is only one Car configuration in LH2 and thus only one molecule per alpha/beta-heterodimer.     

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.     

Structural analysis of the reaction center light-harvesting complex I photosynthetic core complex of Rhodospirillum rubrum using atomic force microscopy

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.     

High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.     

Integration of endocannabinoid and leptin signaling in an appetite-related neural circuit

Recently developed therapeutics for obesity, targeted against cannabinoid receptors, result in decreased appetite and sustained weight loss. Prior studies have demonstrated CB1 receptors (CB1Rs) and leptin modulation of cannabinoid synthesis in hypothalamic neurons. Here, we show that depolarization of perifornical lateral hypothalamus (LH) neurons elicits a CB1R-mediated suppression of inhibition in local circuits thought to be involved in appetite and "natural reward." The depolarization-induced decrease in inhibitory tone to LH neurons is blocked by leptin. Leptin inhibits voltage-gated calcium channels in LH neurons via the activation of janus kinase 2 (JAK2) and of mitogen-activated protein kinase (MAPK). Leptin-deficient mice are characterized by both an increase in steady-state voltage-gated calcium currents in LH neurons and a CB1R-mediated depolarization-induced suppression of inhibition that is 6-fold longer than that in littermate controls. Our data provide direct electrophysiological support for the involvement of endocannabinoids and leptin as modulators of hypothalamic circuits underlying motivational aspects of feeding behavior.     

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.     

Flexibility and size heterogeneity of the LH1 light harvesting complex revealed by atomic force microscopy: functional significance for bacterial photosynthesis

Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q(B) site of the RC to the quinone pool prior to reduction of the cytochrome bc(1) complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (alpha(1)beta(1)Bchl(2)) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.     

Activation of adenylyl cyclase by thienopyrimidine derivatives in rat testes and ovaries

An important role in functioning of reproductive tissues is played by signaling systems that are regulated by luteinizing hormone (LH) and human chorionic gonadotropin (hCG) that include LH receptors as sensor components. Use of LH and hCG in medicine for treatment of diseases of the reproductive system and for solution of tasks of auxiliary reproductive technologies is restricted by their high cost, the necessity of parenteral introduction, and the presence of side effects. However, in the last few years, low molecular weight agonists of LH receptor have been developed that are devoid of these shortcomings and can be administered perorally. The most effective of them are thienopyrimidine derivatives, in particular the Org 43553 compound. The goal of this work was to study the effect of newly synthesized analogs of Org 43553, 5-amino-N-(tert-butyl)-4-(3-(isonicotinamide)phenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (compound 1) and 5-amino-N-(tert-butyl)-2-(methylthio)-4-(3-(thiophene-3-carboxamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (compound 2), on the basal and LH-stimulated adenylyl cyclase (AC) activity in the plasma-membrane fractions isolated from testes and ovaries of rats. Compounds 1 and 2 have been shown to stimulate in a dose-dependent manner the basal AC activity in the membranes isolated from the testes and ovaries, compound 2 being more effective as compared with compound 1. The EC₅₀ values for compounds 1 and 2 on AC activity were 1.05–1.12 and 0.28–0.37 μM, respectively. The stimulating effect of hCG on AC activity was retained in the presence of the thienopyrimidine derivatives, while at low, nonsaturating hormone concentrations, the additivity of the effects of hCG and of compounds 1 and 2 on the AC activity was observed. This indicates that the thienopyrimidine derivatives interact with the allosteric site located in the LH receptor transmembrane channel and does not overlap with the binding site of gonadotropins located in the N-terminal ectodomain. The action of compounds 1 and 2 was tissue-specific and not found in tissues with no LH receptors present. Our obtained data indicate that compounds 1 and 2 may become prototypes for creation of drugs that are regulators of functions of the male and female reproductive systems.     

Isolation,size estimates,and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides

A series of light-harvesting 1 (LH1) complexes was isolated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C from Rhodobacter sphaeroides M21, which lacks the peripheral light-harvesting 2 (LH2) complex. This ladder of LH1 bands was also demonstrated in the wild type, partially superimposed upon a smaller number of LH2 complexes. An assessment of electrophoretic mobility vs acrylamide concentration, in which the reaction center LM particle and annular LH1 and LH2 complexes were used as standards of known structure, indicated that the LH1 gel bands 2 to 10 represent regular oligomers of an alpha beta heterodimeric unit, that vary in size from (alpha beta)(2-3) to (alpha beta)(10-11). The isolated LH1 complexes exhibited oligomeric state dependent optical properties, characterized by red shifts in near-IR absorption and emission maxima at 77 K of approximately 6 nm as aggregate sizes increased from approximately 3 to 7-8 alpha beta-heterodimers, accompanied by shifts in highly polarized fluorescence from the blue to the red side of the absorption band. This has been explained by the oligomerization of heterodimers to form a curvilinear array of excitonically coupled chromophores, with the anisotropic long-wavelength component, designated originally as B896, corresponding to low energy excitonic transitions arising from interactions within inhomogeneous BChl clusters [Westerhuis et al. (1999) J. Phys. Chem. B 103, 7733-7742]. Differences in electrophoretic profiles of LH1 bands between strains M21 and M2192, an LH1-only strain that also lacks PufX, further suggested that the more rapidly migrating bands represent arced fragments of the curvilinear array of LH1 complexes thought to exist as a large closed circular structure only in the latter strain. The electrophoretic banding pattern also indicated that the LH1 complex may be located at the peripheries of dimeric intramembrane particle arrays seen in freeze-fracture replicas of tubular M21 membranes; the possible role for the PufX protein in the assembly of these structures is discussed.     

Solution structures of the core light-harvesting alpha and beta polypeptides from Rhodospirillum rubrum: implications for the pigment-protein and protein-protein interactions

We have determined the solution structures of the core light-harvesting (LH1) alpha and beta-polypeptides from wild-type purple photosynthetic bacterium Rhodospirillum rubrum using multidimensional NMR spectroscopy. The two polypeptides form stable alpha helices in organic solution. The structure of alpha-polypeptide consists of a long helix of 32 amino acid residues over the central transmembrane domain and a short helical segment at the N terminus that is followed by a three-residue loop. Pigment-coordinating histidine residue (His29) in the alpha-polypeptide is located near the middle of the central helix. The structure of beta-polypeptide shows a single helix of 32 amino acid residues in the membrane-spanning region with the pigment-coordinating histidine residue (His38) at a position close to the C-terminal end of the helix. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R.rubrum LH1 alpha and beta-polypeptides are different from those previously reported for the LH1 beta-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 alpha and beta-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 alpha and beta-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2-Gln6) of alpha-polypeptide and a GxxxG motif in the beta-polypeptide.     

Crystallization and preliminary x-ray investigation of purine-nucleoside phosphorylase from Escherichia coli

Crystals of purine-nucleoside phosphorylase from Escherichia coli have been grown from solutions of ammonium sulfate. The crystals are hexagonal with space group P6(1)22 or P6(5)22; the axes are alpha = 106.5 A and c = 241.3 A. The crystals are moderately stable to x-rays and diffract beyond 3.0-A resolution. It appears that the molecule, which is a hexamer, utilizes the 2-fold symmetry of the space group, resulting in three subunits/asymmetric unit.     

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Gout occurs in individuals with hyperuricemia when monosodium urate (MSU) crystals precipitate in tissues and induce acute inflammation via phagocytic cells such as monocytes. MSU crystals have been demonstrated in skin diseases such as tophaceous gout or psoriasis; however, the importance of MSU crystals in the skin is totally unknown. In this study, we found that MSU crystals, through P2Y(6) receptors, stimulated normal human keratinocytes (NHK) to produce IL-1α, IL-8/CXCL8, and IL-6. P2Y(6) receptor expression increased in MSU-stimulated NHK. Both P2Y(6)-specific antagonist and P2Y(6) antisense oligonucleotides significantly inhibited the production of IL-1α, IL-8/CXCL8, and IL-6 by NHK. Similarly, the P2Y(6)-specific antagonist completely inhibited the MSU-induced production of IL-1β by THP-1 cells, a human monocytic cell line. Remarkably, the P2Y(6)-specific antagonist significantly reduced neutrophil influx in both mouse air pouch and peritonitis models. Thus, these results indicate that the P2Y(6) receptor signaling pathway may be a potential therapeutic target for MSU-associated inflammatory diseases, such as tophaceous gout.     

ARF6: a newly appreciated player in G protein-coupled receptor desensitization

The luteinizing hormone/choriogonadotropin hormone receptor (LH/CG R) signals to regulate ovulation, corpus luteum formation, and fetal survival during pregnancy. Agonist binding to the LH/CG R is poorly reversible, emphasizing the importance of a cellular mechanism to temper signaling by a potentially persistently active receptor. Like other G protein-coupled receptors (GPCRs), signaling by this receptor is modulated by its binding of an arrestin. We have identified ADP ribosylation factor 6 (ARF6) as a protein whose activation state is regulated by the LH/CG R and which functions to regulate the availability of plasma membrane-docked arrestin 2 to this receptor. We hypothesize that ARF6 might also serve GPCRs other than the LH/CG R to regulate the availability of arrestin 2 for receptor desensitization.     

Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon.

Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon.