Abstract
Background: UDP-glucuronosyltransferases (UGTs) play a crucial role in maintaining endobiotic homeostasis and metabolizing xenobiotic compounds, particularly clinical drugs. However, the detailed catalytic mechanism of UGTs has not been fully elucidated due to the limited availability of reliable protein structures. Determining the catalytic domain of human UGTs has proven to be a significant challenge, primarily due to the difficulty in purifying and crystallizing the full-length protein.
Objectives: This study focused on the human UGT2B10 C-terminal cofactor binding domain, aiming to provide structural insights into the fundamental catalytic mechanisms.
Methods: In this study, the C-terminal sugar-donor binding domain of human UGT2B10 was purified and crystallized using the vapor-diffusion method. The resulting UGT2B10 CTD crystals displayed high-quality diffraction patterns, allowing for data collection at an impressive resolution of 1.53 Å using synchrotron radiation. Subsequently, the structure of the UGT2B10 CTD was determined using the molecule replacement method with a homologous structure.
Results: The crystals were monoclinic, belonging to the space C2 with unit-cell parameters a = 85.90 Å, b = 58.39 Å, c = 68.87 Å, α = γ = 90°, and β = 98.138°. The Matthews coefficient VM was determined to be 2.24 Å3 Da-1 (solvent content 46.43%) with two molecules in the asymmetric unit.
Conclusion: The crystal structure of UGT2B10 CTD was solved at a high resolution of 1.53 Å, revealing a conserved cofactor binding pocket. This is the first study determining the C-terminal cofactor binding domain of human UGT2B10, which plays a key role in additive drug metabolism.
Graphical Abstract
[1]
Jarrar, Y.; Lee, S.J. The functionality of UDP-glucuro- nosyltransferase genetic variants and their association with drug responses and human diseases. J. Pers. Med., 2021, 11(6), 554.
[http://dx.doi.org/10.3390/jpm11060554] [PMID: 34198586]
[http://dx.doi.org/10.3390/jpm11060554] [PMID: 34198586]
[2]
Dimunová, D.; Matoušková, P.; Podlipná, R.; Boušová, I.; Skálová, L. The role of UDP-glycosyltransferases in xenobio- ticresistance. Drug Metab. Rev., 2022, 54(3), 282-298.
[http://dx.doi.org/10.1080/03602532.2022.2083632] [PMID: 35635097]
[http://dx.doi.org/10.1080/03602532.2022.2083632] [PMID: 35635097]
[3]
Hu, D.G.; Mackenzie, P.I.; Hulin, J.A.; McKinnon, R.A.; Meech, R. Regulation of human UDP-glycosyltransferase (UGT) genes by miRNAs. Drug Metab. Rev., 2022, 54(2), 120-140.
[http://dx.doi.org/10.1080/03602532.2022.2048846] [PMID: 35275773]
[http://dx.doi.org/10.1080/03602532.2022.2048846] [PMID: 35275773]
[4]
Miners, J.O.; Rowland, A.; Novak, J.J.; Lapham, K.; Goosen, T.C. Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucurono- syltransferase reaction phenotyping. Pharmacol. Ther., 2021, 218, 107689.
[http://dx.doi.org/10.1016/j.pharmthera.2020.107689] [PMID: 32980440]
[http://dx.doi.org/10.1016/j.pharmthera.2020.107689] [PMID: 32980440]
[5]
Oda, S.; Fukami, T.; Yokoi, T.; Nakajima, M. A comprehensive review of UDP-glucuronosyltransferase and esterases for drug development. Drug Metab. Pharmacokinet., 2015, 30(1), 30-51.
[http://dx.doi.org/10.1016/j.dmpk.2014.12.001] [PMID: 25760529]
[http://dx.doi.org/10.1016/j.dmpk.2014.12.001] [PMID: 25760529]
[6]
Williams, J.A.; Hyland, R.; Jones, B.C.; Smith, D.A.; Hurst, S.; Goosen, T.C.; Peterkin, V.; Koup, J.R.; Ball, S.E. Drug-drug interactions for UDP-glucuronosyltransferase substrates: A pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab. Dispos., 2004, 32(11), 1201-1208.
[http://dx.doi.org/10.1124/dmd.104.000794] [PMID: 15304429]
[http://dx.doi.org/10.1124/dmd.104.000794] [PMID: 15304429]
[7]
Stingl, J.C.; Bartels, H.; Viviani, R.; Lehmann, M.L.; Brockmöller, J. Relevance of UDP-glucuronosyltransferase polymorphisms for drug dosing: A quantitative systematic review. Pharmacol. Ther., 2014, 141(1), 92-116.
[http://dx.doi.org/10.1016/j.pharmthera.2013.09.002] [PMID: 24076267]
[http://dx.doi.org/10.1016/j.pharmthera.2013.09.002] [PMID: 24076267]
[8]
Rowland, A.; Miners, J.O.; Mackenzie, P.I. The UDP-glucuro- nosyltransferases: Their role in drug metabolism and detoxification. Int. J. Biochem. Cell Biol., 2013, 45(6), 1121-1132.
[http://dx.doi.org/10.1016/j.biocel.2013.02.019] [PMID: 23500526]
[http://dx.doi.org/10.1016/j.biocel.2013.02.019] [PMID: 23500526]
[9]
Yang, N.; Sun, R.; Liao, X.; Aa, J.; Wang, G. UDP-glucurono- syltransferases (UGTs) and their related metabolic cross-talk with internal homeostasis: A systematic review of UGT isoforms for precision medicine. Pharmacol. Res., 2017, 121, 169-183.
[http://dx.doi.org/10.1016/j.phrs.2017.05.001] [PMID: 28479371]
[http://dx.doi.org/10.1016/j.phrs.2017.05.001] [PMID: 28479371]
[10]
Dang, N.L.; Hughes, T.B.; Krishnamurthy, V.; Swamidass, S.J. A simple model predicts UGT-mediated metabolism. Bioinformatics, 2016, 32(20), 3183-3189.
[http://dx.doi.org/10.1093/bioinformatics/btw350] [PMID: 27324196]
[http://dx.doi.org/10.1093/bioinformatics/btw350] [PMID: 27324196]
[11]
Chen, X.; Deng, X.; Zhang, Y.; Wu, Y.; Yang, K.; Li, Q.; Wang, J.; Yao, W.; Tong, J.; Xie, T.; Hou, S.; Yao, J. Computational design and crystal structure of a highly efficient benzoylecgonine hydrolase. Angew. Chem. Int. Ed., 2021, 60(40), 21959-21965.
[http://dx.doi.org/10.1002/anie.202108559] [PMID: 34351032]
[http://dx.doi.org/10.1002/anie.202108559] [PMID: 34351032]
[12]
Yao, J.; Chen, X.; Zheng, F.; Zhan, C.G. Catalytic reaction mechanism for drug metabolism in human carboxylesterase-1: Cocaine hydrolysis pathway. Mol. Pharm., 2018, 15(9), 3871-3880.
[http://dx.doi.org/10.1021/acs.molpharmaceut.8b00354] [PMID: 30095924]
[http://dx.doi.org/10.1021/acs.molpharmaceut.8b00354] [PMID: 30095924]
[13]
Pan, Y.; Gao, D.; Yang, W.; Cho, H.; Yang, G.; Tai, H.H.; Zhan, C.G. Computational redesign of human butyrylcholinesterase for anticocaine medication. Proc. Natl. Acad. Sci., 2005, 102(46), 16656-16661.
[http://dx.doi.org/10.1073/pnas.0507332102] [PMID: 16275916]
[http://dx.doi.org/10.1073/pnas.0507332102] [PMID: 16275916]
[14]
Miners, J.O.; Smith, P.A.; Sorich, M.J.; McKinnon, R.A.; Mackenzie, P.I. Predicting human drug glucuronidation parameters: Application of in vitro and in silico modeling approaches. Annu. Rev. Pharmacol. Toxicol., 2004, 44(1), 1-25.
[http://dx.doi.org/10.1146/annurev.pharmtox.44.101802.121546] [PMID: 14744236]
[http://dx.doi.org/10.1146/annurev.pharmtox.44.101802.121546] [PMID: 14744236]
[15]
Miners, J.O.; Mackenzie, P.I.; Knights, K.M. The prediction of drug-glucuronidation parameters in humans: UDP-glucuro- nosyltransferase enzyme-selective substrate and inhibitor probes for reaction phenotyping and in vitro–in vivo extrapolation of drug clearance and drug-drug interaction potential. Drug Metab. Rev., 2010, 42(1), 196-208.
[http://dx.doi.org/10.3109/03602530903210716] [PMID: 19795925]
[http://dx.doi.org/10.3109/03602530903210716] [PMID: 19795925]
[16]
Operaña, T.N.; Tukey, R.H. Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation. J. Biol. Chem., 2007, 282(7), 4821-4829.
[http://dx.doi.org/10.1074/jbc.M609417200] [PMID: 17179145]
[http://dx.doi.org/10.1074/jbc.M609417200] [PMID: 17179145]
[17]
Radominska-Pandya, A.; Pokrovskaya, I.D.; Xu, J.; Little, J.M.; Jude, A.R.; Kurten, R.C.; Czernik, P.J. Nuclear UDP-glucurono- syltransferases: Identification of UGT2B7 and UGT1A6 in human liver nuclear membranes. Arch. Biochem. Biophys., 2002, 399(1), 37-48.
[http://dx.doi.org/10.1006/abbi.2001.2743] [PMID: 11883901]
[http://dx.doi.org/10.1006/abbi.2001.2743] [PMID: 11883901]
[18]
Fujiwara, R.; Yokoi, T.; Nakajima, M. Structure and protein–protein interactions of human UDP-glucurono- syltransferases. Front. Pharmacol., 2016, 7, 388-403.
[http://dx.doi.org/10.3389/fphar.2016.00388] [PMID: 27822186]
[http://dx.doi.org/10.3389/fphar.2016.00388] [PMID: 27822186]
[19]
Ma, T.; Dai, Y.Q.; Li, N.; Huo, Q.; Li, H.M.; Zhang, Y.X.; Piao, Z.H.; Wu, C.Z. Enzymatic biosynthesis of novel neobavaisoflavone glucosides via Bacillus UDP-glyco- syltransferase. Chin. J. Nat. Med., 2017, 15(4), 281-287.
[http://dx.doi.org/10.1016/S1875-5364(17)30045-6] [PMID: 28527513]
[http://dx.doi.org/10.1016/S1875-5364(17)30045-6] [PMID: 28527513]
[20]
Coutinho, P.M.; Deleury, E.; Davies, G.J.; Henrissat, B. An evolving hierarchical family classification for glycosyl- transferases. J. Mol. Biol., 2003, 328(2), 307-317.
[http://dx.doi.org/10.1016/S0022-2836(03)00307-3] [PMID: 12691742]
[http://dx.doi.org/10.1016/S0022-2836(03)00307-3] [PMID: 12691742]
[21]
Lairson, L.L.; Henrissat, B.; Davies, G.J.; Withers, S.G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem., 2008, 77(1), 521-555.
[http://dx.doi.org/10.1146/annurev.biochem.76.061005.092322] [PMID: 18518825]
[http://dx.doi.org/10.1146/annurev.biochem.76.061005.092322] [PMID: 18518825]
[22]
Bock, K.W. The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal plant arms-race and co-evolution. Biochem. Pharmacol., 2016, 99, 11-17.
[http://dx.doi.org/10.1016/j.bcp.2015.10.001] [PMID: 26453144]
[http://dx.doi.org/10.1016/j.bcp.2015.10.001] [PMID: 26453144]
[23]
Mulichak, A.M.; Lu, W.; Losey, H.C.; Walsh, C.T.; Garavito, R.M. Crystal structure of vancosaminyltransferase GtfD from the vancomycin biosynthetic pathway: interactions with acceptor and nucleotide ligands. Biochemistry, 2004, 43(18), 5170-5180.
[http://dx.doi.org/10.1021/bi036130c] [PMID: 15122882]
[http://dx.doi.org/10.1021/bi036130c] [PMID: 15122882]
[24]
Mulichak, A.M.; Losey, H.C.; Walsh, C.T.; Garavito, R.M. Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics. Structure, 2001, 9(7), 547-557.
[http://dx.doi.org/10.1016/S0969-2126(01)00616-5] [PMID: 11470430]
[http://dx.doi.org/10.1016/S0969-2126(01)00616-5] [PMID: 11470430]
[25]
Miley, M.J.; Zielinska, A.K.; Keenan, J.E.; Bratton, S.M.; Radominska-Pandya, A.; Redinbo, M.R. Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7. J. Mol. Biol., 2007, 369(2), 498-511.
[http://dx.doi.org/10.1016/j.jmb.2007.03.066] [PMID: 17442341]
[http://dx.doi.org/10.1016/j.jmb.2007.03.066] [PMID: 17442341]
[26]
Zhang, L.; Zhu, L.; Qu, W.; Wu, F.; Hu, M.; Xie, W.; Liu, Z.; Wang, C. Insight into tartrate inhibition patterns in vitro and in vivo based on cocrystal structure with UDP-glucurono- syltransferase 2B15. Biochem. Pharmacol., 2020, 172, 113753.
[http://dx.doi.org/10.1016/j.bcp.2019.113753] [PMID: 31837310]
[http://dx.doi.org/10.1016/j.bcp.2019.113753] [PMID: 31837310]
[27]
King, C.; Rios, G.; Green, M.; Tephly, T. UDP-Glucurono- syltransferases. Curr. Drug Metab., 2000, 1(2), 143-161.
[http://dx.doi.org/10.2174/1389200003339171] [PMID: 11465080]
[http://dx.doi.org/10.2174/1389200003339171] [PMID: 11465080]
[28]
Nair, P.C.; Meech, R.; Mackenzie, P.I.; McKinnon, R.A.; Miners, J.O. Insights into the UDP-sugar selectivities of human UDP-glycosyltransferases (UGT): a molecular modeling perspective. Drug Metab. Rev., 2015, 47(3), 335-345.
[PMID: 26289097]
[PMID: 26289097]
[29]
Milani, N.; Qiu, N.; Molitor, B.; Badée, J.; Cruciani, G.; Fowler, S. Use of phenotypically poor metabolizer individual donor human liver microsomes to identify selective substrates of UGT2B10. Drug Metab. Dispos., 2020, 48(3), 176-186.
[http://dx.doi.org/10.1124/dmd.119.089482] [PMID: 31839590]
[http://dx.doi.org/10.1124/dmd.119.089482] [PMID: 31839590]
[30]
Lv, X.; Zhang, J.B.; Hou, J.; Dou, T.Y.; Ge, G.B.; Hu, W.Z.; Yang, L. Chemical probes for human UDP-glucurono- syltransferases: A comprehensive review. Biotechnol. J., 2019, 14(1), 1800002.
[http://dx.doi.org/10.1002/biot.201800002] [PMID: 30192065]
[http://dx.doi.org/10.1002/biot.201800002] [PMID: 30192065]
[31]
Vonrhein, C.; Flensburg, C.; Keller, P.; Sharff, A.; Smart, O.; Paciorek, W.; Womack, T.; Bricogne, G. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr., 2011, 67(4), 293-302.
[http://dx.doi.org/10.1107/S0907444911007773] [PMID: 21460447]
[http://dx.doi.org/10.1107/S0907444911007773] [PMID: 21460447]
[33]
Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.W.; McCoy, A.; McNicholas, S.J.; Murshudov, G.N.; Pannu, N.S.; Potterton, E.A.; Powell, H.R.; Read, R.J.; Vagin, A.; Wilson, K.S. Overview of the CCP 4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr., 2011, 67(4), 235-242.
[http://dx.doi.org/10.1107/S0907444910045749] [PMID: 21460441]
[http://dx.doi.org/10.1107/S0907444910045749] [PMID: 21460441]
[34]
Evans, P.R.; Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D. Biol. Crystallogr., 2013, 69(7), 1204-1214.
[http://dx.doi.org/10.1107/S0907444913000061] [PMID: 23793146]
[http://dx.doi.org/10.1107/S0907444913000061] [PMID: 23793146]
[35]
Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol., 1968, 33(2), 491-497.
[http://dx.doi.org/10.1016/0022-2836(68)90205-2] [PMID: 5700707]
[http://dx.doi.org/10.1016/0022-2836(68)90205-2] [PMID: 5700707]
[36]
Kantardjieff, K.A.; Rupp, B. Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein–nucleic acid complex crystals. Protein Sci., 2003, 12(9), 1865-1871.
[http://dx.doi.org/10.1110/ps.0350503] [PMID: 12930986]
[http://dx.doi.org/10.1110/ps.0350503] [PMID: 12930986]
[37]
McCoy, A.J.; Grosse-Kunstleve, R.W.; Adams, P.D.; Winn, M.D.; Storoni, L.C.; Read, R.J. Phaser crystallographic software. J. Appl. Cryst., 2007, 40(4), 658-674.
[http://dx.doi.org/10.1107/S0021889807021206] [PMID: 19461840]
[http://dx.doi.org/10.1107/S0021889807021206] [PMID: 19461840]
[38]
Adams, P.D.; Afonine, P.V.; Bunkóczi, G.; Chen, V.B.; Davis, I.W.; Echols, N.; Headd, J.J.; Hung, L.W.; Kapral, G.J.; Grosse-Kunstleve, R.W.; McCoy, A.J.; Moriarty, N.W.; Oeffner, R.; Read, R.J.; Richardson, D.C.; Richardson, J.S.; Terwilliger, T.C.; Zwart, P.H. PHENIX : A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr., 2010, 66(2), 213-221.
[http://dx.doi.org/10.1107/S0907444909052925] [PMID: 20124702]
[http://dx.doi.org/10.1107/S0907444909052925] [PMID: 20124702]
[39]
Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr., 2010, 66(4), 486-501.
[http://dx.doi.org/10.1107/S0907444910007493] [PMID: 20383002]
[http://dx.doi.org/10.1107/S0907444910007493] [PMID: 20383002]
[40]
Afonine, P.V.; Grosse-Kunstleve, R.W.; Echols, N.; Headd, J.J.; Moriarty, N.W.; Mustyakimov, M.; Terwilliger, T.C.; Urzhumtsev, A.; Zwart, P.H.; Adams, P.D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D. Biol. Crystallogr., 2012, 68(4), 352-367.
[http://dx.doi.org/10.1107/S0907444912001308] [PMID: 22505256]
[http://dx.doi.org/10.1107/S0907444912001308] [PMID: 22505256]
[41]
Laskowski, R.A.; Macarthur, M.W.; Moss, D.S.; Thornton, J.M. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Cryst., 1993, 26, 283-291.
[http://dx.doi.org/10.1107/S0021889892009944]
[http://dx.doi.org/10.1107/S0021889892009944]
[43]
Lee, S.G.; Salomon, E.; Yu, O.; Jez, J.M. Molecular basis for branched steviol glucoside biosynthesis. Proc. Natl. Acad. Sci., 2019, 116(26), 13131-13136.
[http://dx.doi.org/10.1073/pnas.1902104116] [PMID: 31182573]
[http://dx.doi.org/10.1073/pnas.1902104116] [PMID: 31182573]
Related Journals
Current Protein & Peptide Science
Related Books
Frontiers in Protein and Peptide Sciences
