A Molecular Model of the Human UDP-Glucuronosyltransferase 1A1, Its Membrane Orientation, and the Interactions between Different Parts of the Enzyme

Abstract

The vertebrate UDP-glucuronosyltransferases (UGTs) are membrane-bound enzymes of the endoplasmic reticulum that process both endogenous and exogenous substrates. The human UGTs are well known biologically, but biophysical understanding is scarce, largely because of problems in purification. The one resolved crystal structure covers the C-terminal domain of the human UGT2B7. Here, we present a homology model of the complete monomeric human UGT1A1, the enzyme that catalyzes bilirubin glucuronidation. The enzyme can be seen as composed of four different domains: two large ones, the N-and C-terminal domains, and two small ones, the "envelope" helices and the transmembrane segment that includes the cytoplasmic tail. The hydrophobic core of the N-terminal domain and the two envelope helices that connect the large domains are shown to be structurally well conserved even among distant homologs and can thus be modeled with good certainty according to plant and bacterial structures. We consider alternative solutions for the highly variable N-terminal regions that probably contribute to substrate binding. The bilirubin binding site, known pathological mutations in UGT1A1, and other specific residues have been examined in the context of the model with regard to available experimental data. A putative orientation of the protein relative to the membrane has been derived from the location of predicted N-glycosylation sites. The model presents extensive interactions between the N-and C-terminal domains, the two envelope helices, and the membrane. Together, these interactions could allow for a concerted largescale conformational change during catalysis.

Identification and Functional Characterization of a Novel UDP-Glucuronosyltransferase 2A1 Splice Variant: Potential Importance in Tobacco-Related Cancer Susceptibility

“…UGT2A1 exon 3 is a conserved region throughout all UGT family members (Nagar and Remmel, 2006) and is also highly conserved with the corresponding region of the mouse ortholog UGT2a1 after sequence alignment. The exon 3-encoded protein region of all UGTs is hypothesized to form a portion of the UDPGA cosubstrate binding pocket necessary for UGT function, and similar to the activity results for the splice variant UGT2A1 isoform in the present study, amino acid point mutations in this region have been shown previously to ablate UGT enzyme activity (Miley et al, 2007;Bushey et al, 2011;Laakkonen and Finel, 2010).…”

Three-Dimensional Quantitative Structure-Activity Relationship Studies on UGT1A9-Mediated 3-O-Glucuronidation of Natural Flavonols Using a Pharmacophore-Based Comparative Molecular Field Analysis Model

“…This might indicate that the UGT1A9 protein adopts distinct conformations for aglycone binding and product expelling/releasing, because the turnover is determined in a large part by the departure of product (3-O-glucuronides). This hypothesis is supported by the fact that UGTs undergo dramatic conformation changes during catalysis (Laakkonen and Finel, 2010). Therefore, as mentioned earlier, conventional use of K m or K i as indicators of substrate selectivity might lead to erroneous interpretation of interaction between substrates and UGT1As.…”

Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

“…To understand the role of these 13% residues, we modelled both isoforms using Modeller following the same procedure as that adopted for modelling UGT1A1. [58] Determination of the 3D structure of membranous proteins is a tedious task due to difficulties in crystallisation and their insolubility in normal solvents, resulting in the availability of very few crystal or NMR structures of membrane proteins. As the 3D structure of proteins is one of the crucial steps in the drug discovery process, a series of 3D protein structures important for drug design was developed by homology modelling to acquire the structural information in a timely manner [69,70] and proved very useful.…”

“…The homology modelling of UGT1A9 and UGT1A10 was performed by a methodology similar to that used by Laakkonen and Finel, [58] to model UGT1A1. Only, NTD and CTD were modelled as the focus throughout this study was to analyse the substrate and co-substrate binding sites, and the reported data confirmed that these sites are located at the NTD-CTD interface.…”