Crystal structure of pre-activated arrestin p44.

Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ∼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296-Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.

Crystal structure of metarhodopsin II.

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαßγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 Å and 2.85 Å crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

Crystal structure of the ligand-free G-protein-coupled receptor opsin.

In the G-protein-coupled receptor (GPCR) rhodopsin, the inactivating ligand 11-cis-retinal is bound in the seven-transmembrane helix (TM) bundle and is cis/trans isomerized by light to form active metarhodopsin II. With metarhodopsin II decay, all-trans-retinal is released, and opsin is reloaded with new 11-cis-retinal. Here we present the crystal structure of ligand-free native opsin from bovine retinal rod cells at 2.9 ångström (A) resolution. Compared to rhodopsin, opsin shows prominent structural changes in the conserved E(D)RY and NPxxY(x)(5,6)F regions and in TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 A, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, some of which were attributed to an active GPCR state, reorganize the empty retinal-binding pocket to disclose two openings that may serve the entry and exit of retinal. The opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.

Crystal structure of opsin in its G-protein-interacting conformation.

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.

A G protein-coupled receptor at work: the rhodopsin model.

G protein-coupled receptors (GPCRs) are ubiquitous signal transducers in cell membranes, as well as important drug targets. Interaction with extracellular agonists turns the seven transmembrane helix (7TM) scaffold of a GPCR into a catalyst for GDP and GTP exchange in heterotrimeric Galphabetagamma proteins. Activation of the model GPCR, rhodopsin, is triggered by photoisomerization of its retinal ligand. From the augmentation of biochemical and biophysical studies by recent high-resolution 3D structures, its activation intermediates can now be interpreted as the stepwise engagement of protein domains. Rearrangement of TM5-TM6 opens a crevice at the cytoplasmic side of the receptor into which the C terminus of the Galpha subunit can bind. The Galpha C-terminal helix is used as a transmission rod to the nucleotide binding site. The mechanism relies on dynamic interactions between conserved residues and could therefore be common to other GPCRs.

Opsin, a structural model for olfactory receptors?

Receptor-ligand interaction: Olfactory receptors (ORs) are G-protein-coupled receptors (GPCRs), which detect signaling molecules such as hormones and odorants. The structure of opsin, the GPCR employed in vision, with a detergent molecule bound deep in its orthosteric ligand-binding pocket provides a template for OR homology modeling, thus enabling investigation of the structural basis of the mechanism of odorant-receptor recognition.

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface.

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the alpha-subunit of intracellular G proteins (Galphabetagamma). Kinetic analysis of transducin (G(t)alphabetagamma) activation shows that an intermediary R*xG(t)alphabetagamma.GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*xG protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G(t)alpha C-terminal peptide (GalphaCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal alpha-helix of G(t)alpha (alpha5) and shows how alpha5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them--termed stable or S-interaction--matches the position of the GalphaCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GalphaCT and conserved E(D)RY and NPxxY(x)(5,6)F regions of the GPCR. The alternative fit--termed intermediary or I-interaction--is distinguished by a tilt (42 degrees ) and rotation (90 degrees ) of alpha5 relative to the S-interaction and shows different alpha5 contacts with the NPxxY(x)(5,6)F region and the second cytoplasmic loop of R*. From the 2 alpha5 interactions, we derive a "helix switch" mechanism for the transition of R*xG(t)alphabetagamma.GDP to the nucleotide-free R*xG protein complex that illustrates how alpha5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.

Structural evidence for visual arrestin priming via complexation of phosphoinositols.

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail.

Light-induced conformational changes of the chromophore and the protein in phytochromes: bacterial phytochromes as model systems.

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

Centrins in retinal photoreceptor cells: regulators in the connecting cilium.

Changes in the intracellular Ca2+ concentration regulate the visual signal transduction cascade directly or more often indirectly through Ca2+-binding proteins. Here we focus on centrins, which are members of a highly conserved subgroup of the EF-hand superfamily of Ca2+-binding proteins in photoreceptor cells of the vertebrate retina. Centrins are commonly associated with centrosome-related structures. In mammalian retinal photoreceptor cells, four centrin isoforms are expressed as prominent components in the connecting cilium linking the light-sensitive outer segment compartment with the metabolically active inner segment compartment. Our data indicate that Ca2+-activated centrin isoforms assemble into protein complexes with the visual heterotrimeric G-protein transducin. This interaction of centrins with transducin is mediated by binding to the betagamma-dimer of the heterotrimeric G-protein. More recent findings show that these interactions of centrins with transducin are reciprocally regulated via site-specific phosphorylations mediated by the protein kinase CK2. The assembly of centrin/G-protein complexes is a novel aspect of translocation regulation of signalling proteins in sensory cells, and represents a potential link between molecular trafficking and signal transduction in general.

Calcium-dependent assembly of centrin-G-protein complex in photoreceptor cells.

Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca(2+)-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the betagamma-complex. The Ca(2+)-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.

Insights into functional aspects of centrins from the structure of N-terminally extended mouse centrin 1.

Centrins are members of the family of Ca(2+)-binding EF-hand proteins. In photoreceptor cells, centrin isoform 1 is specifically localized in the non-motile cilium. This connecting cilium links the light-sensitive outer segment with the biosynthetic active inner segment of the photoreceptor cell. All intracellular exchanges between these compartments have to occur through this cilium. Three-dimensional structures of centrins from diverse organisms are known, showing that the EF-hand motifs of the N-terminal domains adopt closed conformations, while the C-terminal EF-hand motifs have open conformations. The crystal structure of an N-terminally extended mouse centrin 1 (MmCen1-L) resembles the overall structure of troponin C in its two Ca(2+) bound form. Within the N-terminal extension in MmCen1-L, residues W24 and R25 bind to the C-terminal domain of centrin 1 in a target-protein-like geometry. Here, we discuss this binding mode in connection with putative interaction sites of the target-protein transducin and the self-assembly of centrins.

Crystallization and preliminary X-ray studies of mouse centrin1.

Centrins belong to a family of Ca2+-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure-function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW approximately 22 240 Da) could be crystallized at room temperature, using 20-25%(w/v) PEG 1500, 5-10%(v/v) ethylene glycol and 1-2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, beta = 109.4 degrees. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 A3 Da(-1).

Serum lipid concentrations correlate more strongly with total body fat than with body mass index in obese humans.

BACKGROUND: To investigate the associations of body mass index (BMI) and total body fat (TBF) vs. blood lipid concentrations, we measured six anthropometric parameters, body fat mass, and serum lipid profiles in 1529 apparently healthy adults. METHODS: TBF was assessed using a body fat analyzer. Serum concentrations of triglyceride, total cholesterol, and low- or high-density lipoprotein cholesterol (LDL-C or HDL-C) were determined by standard enzymatic procedures. RESULTS: Serum lipid concentrations were more strongly correlated with TBF than with BMI or waist circumference in both men and women. The mean concentrations of total cholesterol in the subjects with high fatness (TBF>95th percentile) were significantly higher than those for the subjects with low fatness (TBF<5th percentile; p<0.01), but no significant differences were observed in serum lipid levels between overweights (BMI>95th percentile) and underweights (BMI<5th percentile). The incidence of hypercholesterolemia was significantly higher in the subjects with high fatness (TBF>95th percentile) than the corresponding overweight subjects (BMI>95th percentile; p<0.01). CONCLUSION: TBF is more strongly associated with serum lipid concentrations in adults, at least as compared to BMI.

Transmembrane signaling by GPCRs: insight from rhodopsin and opsin structures.

G-protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors, are the largest family of membrane proteins in the human genome. As versatile signaling molecules, they mediate cellular responses to extracellular signals. Diffusible ligands like hormones and neurotransmitters bind to GPCRs to modulate GPCR activity. An extraordinary and highly specialized GPCR is the photoreceptor rhodopsin which contains the chromophore retinal as its covalently bound ligand. For receptor activation the configuration of retinal is altered by photon absorption. To date, rhodopsin is the only GPCR for which crystal structures of inactive, active and ligand-free conformations are known. Although the photochemical activation is unique to rhodopsin, many mechanistic insights from this receptor can be generalized for GPCRs.

A ligand channel through the G protein coupled receptor opsin.

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B.

Crystallization and preliminary X-ray crystallographic analysis of the N-terminal photosensory module of phytochrome Agp1, a biliverdin-binding photoreceptor from Agrobacterium tumefaciens.

Phytochromes are photochromic photoreceptors with a bilin chromophore that have been found in plants and bacteria. Typical bacterial phytochromes are composed of an N-terminal photosensory chromophore module and a C-terminal protein kinase. The former contains the chromophore, which allows phytochromes to adopt the two interconvertible spectral forms, Pr and Pfr. The N-terminal photosensory module of Agrobacterium phytochrome Agp1, Agp1-M15, was used for crystallization studies. The protein was either assembled with the natural chromophore biliverdin or a sterically locked synthetic biliverdin-derivative, termed 15Za. The last-named adduct does not undergo photoisomerization due to an additional carbon chain between the rings C and D of the chromophore. Both adducts could be crystallized, but the resolution was largely improved by the use of 15Za. Crystals of biliverdin-Agp1-M15 diffract to 6A resolution and belong to the tetragonal space group I422 with unit cell dimensions a = b = 171 Angstroms, c = 81 Angstroms, crystals of 15Za-Agp1-M15 belong to the same space group with similar unit cell dimensions a = b = 174 Angstroms, c = 80 Angstroms, but diffract to 3.4 Angstroms resolution. Assuming the asymmetric unit to be occupied by one monomer of 55kDa, the unit cell contains 54-55% solvent with a crystal volume per protein mass, V(m), of 2.7 Angstroms(3) Da(-1).

Crystallization and preliminary X-ray diffraction studies of alpha-cyclodextrin glucanotransferase isolated from Bacillus macerans.

Single crystals of alpha-cyclodextrin glucanotransferase isolated from Bacillus macerans have been grown with polyethylene glycol 6000 as a precipitating agent by sitting-drop vapour diffusion at room temperature. The crystals were suitable for X-ray analysis and diffracted to at least 2.0 A (space group P2(1)2(1)2(1)), with unit-cell parameters a = 66.79 (2), b = 79.66 (1), c = 141.16 (1) A. Assuming the asymmetric cell to be occupied by a monomer of 74 kDa, the unit cell contains 42.6% solvent with a crystal Volume per protein mass, V(M), of 2.53 A(3) Da(-1).

Preliminary X-ray characterization of the ribonuclease P (C5 protein) from Escherichia coli: expression, crystallization and cryoconditions.

The gene for Escherichia coli ribonuclease P (RNase P) protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST fusion proteins in E. coli at a high level. After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized with 2.5-2.6 M (NH(4))(2)HPO(4)/(NH(4))H(2)PO(4) pH 7.0 at room temperature. These crystals are suitable for X-ray analysis, belong to the space group P3(1)21 or P3(2)21 (unit-cell parameters a = b = 66.67, c = 142.09 A) and diffract to 2.9 A at 100 K using sorbitol and glycerol as cryoprotectants. For three molecules in the asymmetric unit a V(M) of 2.17 A(3) Da(-1) was calculated.

Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells.

Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity binding to Gtbetagamma and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gbetagamma of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.