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1.  Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutans  
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
doi:10.1107/S1744309110029714
PMCID: PMC2935234  PMID: 20823533
glucansucrase; dental caries; Streptococcus mutans
2.  Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist 
Nature  2012;482(7386):547-551.
The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves.1–5 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiologic control of cardiovascular function through activation of G protein-coupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of antagonist-bound M2 receptor, the first human acetylcholine receptor to be characterized structurally. The antagonist QNB binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all 5 muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The M2 receptor structure provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
doi:10.1038/nature10753
PMCID: PMC3345277  PMID: 22278061
3.  G protein-coupled receptor inactivation by an allosteric inverse-agonist antibody 
Nature  2012;482(7384):237-240.
G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states.1-13 Conformational changes induced by extracellular ligands binding to GPCRs result in a cellular response through the activation of G-proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain.14 In this study, we have successfully raised a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket. The structure of the A2AAR-antibody Fab fragment (Fab2838) complex reveals that the fragment, unexpectedly, recognises the intracellular surface of A2AAR and that its complementarity determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein C-terminal fragment in the active opsin structure1 and to the CDR-3 of the nanobody in the active β2 adrenergic receptor structure2 but locks the A2AAR in an inactive conformation. These results shed light on a novel strategy to modulate GPCR activity.
doi:10.1038/nature10750
PMCID: PMC3303121  PMID: 22286059
4.  Structure of the human histamine H1 receptor complex with doxepin 
Nature  2011;475(7354):65-70.
Summary
The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine-H1 receptor (H1R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of H1R complex with doxepin, a first-generation H1R-antagonist. Doxepin sits deep in the ligand binding pocket and directly interacts with the highly conserved Trp4286.48, a key residue in GPCR activation. This well-conserved pocket with mostly hydrophobic nature contributes to low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H1R-antagonists reveals that the unique carboxyl-group present in this class of compounds interacts with Lys1915.39 and/or Lys179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors defining how minor differences in receptor lead to pronounced selectivity differences with small molecules.
doi:10.1038/nature10236
PMCID: PMC3131495  PMID: 21697825
5.  Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae 
Background
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.
Results
We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.
Conclusions
We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
doi:10.1186/1475-2859-11-78
PMCID: PMC3495400  PMID: 22694812
G-protein coupled receptor; Membrane protein; High expression; Screening; Receptor variants; Structural study; Saccharomyces cerevisiae
6.  Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis 
Background
Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.
Results
The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.
Conclusion
Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.
doi:10.1186/1475-2859-10-24
PMCID: PMC3094209  PMID: 21513509
7.  Characterization of the β-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16* 
The Journal of Biological Chemistry  2010;285(36):28373-28378.
G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.
doi:10.1074/jbc.M110.144444
PMCID: PMC2934701  PMID: 20605788
Calcium Imaging; G-protein-coupled Receptors (GPCR); Membrane Proteins; Receptor Structure-Function; Signal Transduction; Bitter; Taste Receptor; Binding Site; Molecular Dynamics; Molecular Modeling
8.  Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells 
PLoS ONE  2009;4(11):e7827.
Background
Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood.
Methodology/Principal Findings
Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed.
Conclusions and Significance
These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.
doi:10.1371/journal.pone.0007827
PMCID: PMC2772020  PMID: 19915671
9.  Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research 
PLoS ONE  2008;3(12):e3822.
Background
Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.
Methodology/Principal Findings
Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.
Conclusions and Significance
The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.
doi:10.1371/journal.pone.0003822
PMCID: PMC2585475  PMID: 19048102
10.  Roles of thromboxane A2 and prostacyclin in the development of atherosclerosis in apoE-deficient mice 
Journal of Clinical Investigation  2004;114(6):784-794.
Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE–/–TP–/– mice exhibited a significant delay in atherogenesis, and apoE–/–IP–/– mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE–/–IP–/– mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE–/–TP–/– mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE–/–IP–/– and apoE–/–TP–/– mice, respectively, than in those of apoE–/– mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE–/–IP–/– mice than in either apoE–/–TP–/– or apoE–/– mice. We conclude that TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.
doi:10.1172/JCI200421446
PMCID: PMC516261  PMID: 15372102
11.  The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut 
We used mice deficient in each of the eight types and subtypes of prostanoid receptors and examined the roles of prostanoids in dextran sodium sulfate–induced (DSS-induced) colitis. Among the prostanoid receptor–deficient mice, only EP4-deficient mice and not mice deficient in either DP, EP1, EP2, EP3, FP, IP, or TP developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. This phenotype was mimicked in wild-type mice by administration of an EP4-selective antagonist (AE3-208). The EP4 deficiency impaired mucosal barrier function and induced epithelial loss, crypt damage, and aggregation of neutrophils and lymphocytes in the colon. Conversely, administration of an EP4-selective agonist (AE1-734) to wild-type mice ameliorated severe colitis normally induced with 7% DSS, while that of AE3-208 suppressed recovery from colitis and induced significant proliferation of CD4+ T cells. In vitro AE3-208 enhanced and AE1-734 suppressed the proliferation and Th1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP4-deficient mice. We conclude that EP4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response.
doi:10.1172/JCI14459
PMCID: PMC150928  PMID: 11927615

Results 1-11 (11)