Analysis of Opioid Binding to UDP-Glucuronosyltransferase 2B7 Fusion Proteins Using Nuclear Magnetic Resonance Spectroscopy
- Birgit L. Coffman1,
- William R. Kearney2,
- Mitchell D. Green1,
- Robert G. Lowery3 and
- Thomas R. Tephly1
- 1Department of Pharmacology (B.L.C., M.D.G., T.R.T.) and 2Nuclear Magnetic Resonance Facility (W.R.K), College of Medicine, University of Iowa, Iowa City, Iowa; and 3PanVera Corp. Madison, Wisconsin (R.G.L)
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 ofd-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 μ-receptor and to cytochrome 2D6, are proposed.
Footnotes
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Send reprint requests to: Dr. Thomas R. Tephly, University of Iowa, Department of Pharmacology, BSB 2-452, Iowa City, IA 52242. E-mail: ttephly{at}blue.weeg.uiowa.edu
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This work was supported by National Institutes of Health Grant GM26221 (to T.R.T.) and National Institutes of Health Grant R43 GM59542 (to R.G.L.). Support from the College of Medicine, University of Iowa, is also acknowledged (to W.R.K.).
- Abbreviations:
- UGT
- UDP-glucuronosyltransferase
- UDPGlcUA
- UDP-glucuronic acid
- AZT
- zidovudine
- MBP
- maltose binding protein
- CD
- circular dichroism
- NOESY
- nuclear Overhauser effect spectroscopy
- TRNOESY
- transferred nuclear Overhauser effect spectroscopy
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- Received December 2, 2000.
- Accepted March 2, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
