Homology Modeling of Human Uridine-5'-diphosphate-glucuronosyltransferase 1A6 Reveals Insights into Factors Influencing Substrate and Cosubstrate Binding

Abstract

The elimination of numerous endogenous compounds and xenobiotics via glucuronidation by uridine-5'-diphosphate glycosyltransferase enzymes (UGTs) is an essential process of the body's chemical defense system. UGTs have distinct but overlapping substrate preferences, but the molecular basis for their substrate specificity remains poorly understood. Three-dimensional protein structures can greatly enhance our understanding of the interactions between enzymes and their substrates, but because of the inherent difficulties in purifying and crystallizing integral endoplasmic reticulum membrane proteins, no complete mammalian UGT structure has yet been produced. To address this problem, we have created a homology model of UGT1A6 using I-TASSER to explore, in detail, the interactions of human UGT1A6 with its substrates. Ligands were docked into our model in the presence of the cosubstrate uridine-5'-diphosphate-glucuronic acid, interacting residues were examined, and poses were compared to those cocrystallized with various plant and bacterial glycosyltransferases (GTs). Our model structurally resembles other GTs, and docking experiments replicated many of the expected UGT-substrate interactions. Some bias toward the template structures' protein-substrate interactions and binding preferences was evident.

Figure 1

Structure of UGT1A6. (A) Final refined homology model of UGT1A6. Helices are shown as spirals, and β-sheets are shown as broad ribbons. The C-terminal domain (blue) is shown at the top, with the N-terminal domain (green) shown at the bottom of the image. The envelope helices (magenta) are shown to the left, with the TM domain (purple) at the bottom. The putative dimerization domain (PDPVSYIPRCY) of UGT1A6 is highlighted in orange. (B) Schematic diagram of the UGT1A6 structure. Colors are the same as in (A), with α-helices shown as cylinders and β-sheets shown as arrows. The predicted Nα2 and Nα6 helices were not present in the final model and have been rendered as transparent cylinders in the diagram as a reference as to where they would be expected to form.

Figure 2

Comparison of initial I-TASSER generated homology models. The models generated without the use of the 2B7 crystal as a guide are shown in teal (without the signal peptide, NSNT) and green (with the signal peptide, WSNT). The models generated using the 2B7 structure as a guide are shown in blue (with the signal peptide, WSWT) and purple (without the signal peptide, NSWT). (A) Key catalytic residues H38 and D150 in the NSNT and NSWT models differ by only a minute amount between the models. (B) Nα2 helix is not present in the NSWT and WSWT models but does appear in the NSNT and WSNT models. (C) Orientation, length, and positioning of the Nα3 helices differed substantially between models. (D) Nα5 helices also were substantially different between models, with the Nα5-2 positions of the NSWT/WSWT and NSNT/WSNT in different locations and orientations, and the Nα5-3 helix shifted down in the NSNT/WSNT models compared to the NSWT/WSWT model, as demonstrated by the position of the highlighted F226 residue (yellow).

Figure 3

UDPGA interacts with multiple residues in the C-terminal domain as well as three residues in the N-terminal domain (S37, R172, and R256). (A) UDPGA docked into the cleft located between the C-terminal (blue ribbons) and N-terminal domains with several interacting residues indicated. (B) 2D schematic diagram of all non-van der Waals UDPGA–UGT1A6 interactions. Classical hydrogen bonds are shown in dark green, nonclassical hydrogen bonds, in which the donor is a polarized carbon atom, are shown in pale green, electrostatic interactions are shown in orange, hydrophobic interactions in pink, with π–π stacking interactions in dark pink and π–alkyl interactions in light pink, and unfavorable donor–donor interactions are shown in red.

Figure 4

Top scoring compounds were all small flavonoids with a limited number of direct interactions. (A) 3D view of quercetin docked into the receptor (-CDOCKER energy: 73.3885 kcal mol^–1), (B) kaempferol (72.648 kcal mol^–1), (C) apigenin (64.9334 kcal mol^–1), (D) prunetin (61.2897 kcal mol^–1), (E) galangin (60.0664 kcal mol^–1). Interaction colors are as shown in Figure 3.

Figure 5

Comparison of three docked substrates and the same substrates cocrystallized in plant GTs. Residue-substrate interactions are colored as shown in Figure 3. (A) Quercetin (QUE) docked with UDPGA in UGT1A6 (B) Quercetin cocrystallized with UDP in the V. vinifera flavonoid 3-O glucosyltransferase UFGT (PDB: 2C9Z). (C) Chemical structure of quercetin/kaempferol with common glucuronidation sites indicated with arrows. The circled 3′-OH group is absent in kaempferol. (D) Kaempferol (KMP) docked into UGT1A6 with UDPGA. (E) Kaempferol cocrystallized with U2F in the V. vinifera UFGT (PDB: 2C1Z). F. Kaempferol crystallized in the Clitoria ternatea anthocyanidin GT (PDB: 4REL) in the absence of a cosubstrate. (G) TCP docked in UDPGA. (H) TCP cocrystallized with U2F in the Arabidopsis thaliana hydroquinone glucosyltransferase UGT72B1 (PDB: 2VCE). (I) Chemical structure of TCP with the glucuronidation site indicated with an arrow.

Figure 6

Comparison of the average K_m equivalent (μM) with the -CDOCKER score for 13 compounds with available enzyme kinetics data. There was no correlation between the enzyme kinetics data and -CDOCKER energy scores for these compounds, 4-MU, 4-methylumbelliferone.

Figure 7

rmsd of the UGT1A6 model and bound cosubstrate and substrate over the course of the molecular dynamics simulation. The UGT1A6 protein backbone rmsd is shown in black, UPDGA in blue, and quercetin in pink.