Stability And Dimerization Mechanism Of Human UDP-Glucuronosyltransferase(UGT) 2B7

The human uridine diphosphate glucuronosyltransferases(UGTs)are crucial phase Ⅱ metabolism enzymes that detoxify a large amount of essential endobiotics and xenobiotics,by catalyzing the transfer of the glucuronic acid group from uridine diphosphate glucuronic acid(UDPGA)to a specific substrate(also known as aglycone).The major nucleophilic chemical groups of specific substrates include hydroxyl,carboxyl,amino,and sulfhydryl.The UGT enzymes have been classified in two major families in humans,namely UGT1 A and UGT2(subdivided in UGT2 A and UGT2B).The human UGTs are classic type Ⅰ transmembrane glycoproteins,mainly localized in the endoplasmic reticulum(ER)membrane,consisting of approximately 530 amino acids.UGTs have two domains,the N-terminal domain(NTD)with a cleavable signal peptide accountable for targeting the ER membrane and the C-terminal transmembrane domain(CTD)anchoring the protein to the ER membrane.At the CTD,a single transmembrane helix(TH)contains about 20 residues that transmits the phospholipid bilayer from lumen to the cytosol.The flexible single transmembrane helix makes the three-dimensional structures of full-length human UGTs still undetermined,which limits the elucidation of the interaction mechanisms between UGTs and different substrates.In addition,several studies also show that UGTs can be homodimerized or heterodimerized and act as dimers in monoglucuronide formation or as tetramers in diglucuronide formation.Co-localization and protein–protein interactions between UGTs allows concerted metabolism to occur more efficiently.Understanding the dimerization mechanisms of UGTs are critical for predicting the drug-drug interactions,preventing the drug toxicity,and implementing the precision treatment.UGT2B7 is one of the most important UGTs that glucuronidates abundant endobiotics and xenobiotics,such as morphine,zidovudine,estriol,and anticancer drugs.In this study,we focused on the expression and purification of recombinant UGT2B7 by optimizing the insertion sites for the thermostabilized fusion protein apocytochrome b562RIL(BRIL)and various mutations to improve the protein yields and homogeneity.Preparation of the recombinant proteins with high purity accelerated the measurement of pharmacokinetic parameters of UGT2B7.Moreover,the dissociation constants(KD)of two classical substrates(zidovudine and androsterone)and two inhibitors(schisanhenol and hesperetin)of UGT2B7 were determined using the surface plasmon resonance spectroscopy(SPR)for the first time.Furthermore,we examined the thermostability and enzyme kinetics of the recombinant UGT2B7 proteins.The methods described in this study could be broadly applied to other UGTs and are expected to provide the basis for the exploration of metabolic enzyme kinetics,the mechanisms of drug metabolisms and drug interactions,changes in pharmacokinetics,and pharmacodynamics studies in vitro.UGT2B7 has been observed to form homo-or heterodimers that affect its enzymatic activities.In this study,we investigated protein-protein interactions among UGT2B7 and other metabolism enzymes,by performing a systematic quantitative fluorescence resonance energy transfer(FRET)analysis in combination with coimmunoprecipitation(Co-IP)assay.The different UGT1A9 and UGT2B7 allozyme dimers showed distinct differences in FRET efficiencies and donor-acceptor distances,indicating differences in interaction ability.In addition,the glucuronidation activities of heterodimers were further tested with zidovudine as the substrate of UGT2B7 and with propofol as the substrate of UGT1A9.After systematically screened,we obtained stable UGT1A9-UGT2B7 heterodimer.These results provide insights into in-depth understanding of UGT1A9-UGT2B7 heterodimer and the association between dimerization and glucuronidation activity.In the study of protein interaction interface,we introduced various methods to stabilize heterodimer proteins,such as Nanodiscs,Nano Luc Binary technology(Nano Bi T),and cross-linking technology.Additionally,we optimized the expression and purification condition on various constructs and successfully obtained quality proteins,which were used to determinate the three-dimensional structure with single particle cryo-electron microscopy.We assessed the amino acid interactions at the dimer interface and found that the arginine at 210 and leucine at 270 in UGT2B7 had a significant effect on the stability of heterodimer.The obtained evidences provide insights into the combined effects of polymorphisms and protein–protein interactions on UGT1A9 and UGT2B7 catalytic activity in vitro,which allow understanding of UGTs dimerization and changing in metabolite formation.