Analysis of opioid binding to UDP-glucuronosyltransferase 2B7 fusion proteins using nuclear magnetic resonance spectroscopy

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform th at catalyzes the conjugation of many endogenous and exogenous compounds, am ong 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 fusi on proteins, 2B7F1 and 2B7F2, incorporating the first 157 or 119 amino acid s, respectively, of UGT2B7 were expressed in Escherichia coil and purified by affinity chromatography. NMR spectroscopy using one-dimensional spectra, the inversion recovery method, and the transferred nuclear Overhauser effe ct 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 fir st 119 amino acids and to undergo a conformational change upon binding, as demonstrated by transferred nuclear Overhauser effect spectroscopy. Dissoci ation constants were obtained for morphine, naloxone, buprenorphine, and zi dovudine, and the results were confirmed by equilibrium dialysis determinat ions. Two possible opioid binding sites, based on the nearest neighbors fro m opioid binding to the mu -receptor and to cytochrome 2D6, are proposed.