title: Analysis of opioid binding to UDP-glucuronosyltransferase 2B7 fusion proteins using nuclear magnetic resonance spectroscopy.
abstract: The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. In this study, we demonstrate that the opioid binding site in UGT2B7 is within the first 119 amino-terminal amino acids. Two maltose binding protein fusion proteins, 2B7F1 and 2B7F2, incorporating the first 157 or 119 amino acids, respectively, of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR spectroscopy using one-dimensional spectra, the inversion recovery method, and the transferred nuclear Overhauser effect spectroscopy was used to study the binding properties of opioids to the fusion proteins. Morphine was found to bind at a single site within the first 119 amino acids and to undergo a conformational change upon binding, as demonstrated by transferred nuclear Overhauser effect spectroscopy. Dissociation constants were obtained for morphine, naloxone, buprenorphine, and zidovudine, and the results were confirmed by equilibrium dialysis determinations. Two possible opioid binding sites, based on the nearest neighbors from opioid binding to the micro-receptor and to cytochrome 2D6, are proposed.
authors.affiliation: University of Iowa, Department of Pharmacology, College of Medicine, Iowa City, IA 52242, USA.
authors.forename: B L
authors.initials: BL
authors.lastname: Coffman
authors.forename: W R
authors.initials: WR
authors.lastname: Kearney
authors.forename: M D
authors.initials: MD
authors.lastname: Green
authors.forename: R G
authors.initials: RG
authors.lastname: Lowery
authors.forename: T R
authors.initials: TR
authors.lastname: Tephly
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009294:Narcotics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009294:Narcotics; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase