Prediction of the three-dimensional structure for the rat urotensin II receptor, and comparison of the antagonist binding sites and binding selectivity between human and rat receptors from atomistic simulations

Abstract

Urotensin-II (U-II) has been shown to be the most potent mammalian vasoconstrictor known. Thus, a U-II antagonist might be of therapeutic value in a number of cardiovascular disorders. However, interspecies variability of several nonpeptidic ligands complicates the interpretation of in vivo studies of such antagonists in preclinical animal disease models. ACT058362 is a selective antagonist for the human U-II receptor (hUT2R) with a reported K(d) value of approximately 4 nM in a molecular binding assay, but it is reported to bind weakly to rat UT2R (rUT2R), with a K(d) value of approximately 1 500 nM. In contrast, the arylsulphonamide SB706375 is a selective antagonist against both hUT2R (K(d)= approximately 9 nM) and rUT2R (K(d)= approximately 21 nM). To understand the species selectivity of the UT2R, we investigated the binding site of ACT058362 and SB706375 in both hUT2R and rUT2R to explain the dramatically lower (approximately 400-fold) affinity of ACT058362 for rUT2R and the similar affinity (approximately 10 nM) of SB706375 for both UT2Rs. These studies used MembStruk and MSCDock to predict the UT2R structure and the binding site of ACT058362 and SB706375. Based on binding energies, we found two binding modes each with D130(3.32) as the crucial anchoring point (Ballesteros-Weinstein numbering given in superscript). We predict that ACT058362 (an aryl-amine-aryl or ANA ligand) binds in the transmembrane (TM) 3456 region, while SB706375 (an aryl-aryl-amine or AAN ligand) binds in the TM 1237 region. These predicted sites explain the known differences in binding of the ANA ligand to rat and human receptors, while explaining the similar binding of the AAN compound to rat and human receptors. Moreover the predictions explain currently available structure-activity relationship (SAR) data. To further validate the predicted binding sites of these ligands in hUT2R and rUT2R, we propose several mutations that would help define the structural origins of differential responses between UT2R of different species, potentially indicating novel UT2R antagonists with cross-species high affinity.

Figure 1

(Top) The chemical structures of ACT058362 in green and SB-706375 in orange with its hybrid compound in cyan. (Bottom) the superimposition of the lowest E conformers of these compounds through atom-by-atom fitting among three atoms marked with asterisks. The root mean square deviation (RMSD) are 0.63 (for N⁺, N_ar, C_al of ACT-058362 to N⁺, N_ar, C_al of ACT-SB hybrid) and 1.12 Å (for N⁺, S, C_ar of SB-706375 to N⁺, S, C_ar of ACT-SB hybrid).

Figure 2

(Top) The predicted seven transmembrane (TM) regions and (Bottom) the hydropathy prediction from TMPred2nd for rat Urotensin II receptor. Hydrophobic centers marked with asterisks were calculated by the peak method. Highly conserved residues in each TM are underlined.

Figure 3

Pairwise alignment of rat and human Urotensin II receptor (GPCR14). Each transmembrane (TM) helix predicted by TMPredict program is shown with grey shading. Highly conserved residues in Family A receptors are displayed in boxes with Ballesteros numbers. The residues in bold face are important amino acids for UT-II binding. Length= 389 amino acids, Score= 489 bits (1125), Expect=1e-137, Method: Composition-based stats. Identities= 266/328 (74% in whole sequence, 89% in TM helix). Positives= 290/328 (88%), Gaps= 2/328 (0%).

Figure 4

Interhelical interaction energies of MembScream. E-polar energy of each transmembrane (TM) was calculated and plotted radically outward in kcal/mol. In the plot of Scream E, 0 is the lowest Scream E, while the others are the relative E compared with the lowest one. Energetically preferred angles of the rUT2R were 30° of TM4, 60° of TM5, −30° for TM6 and 0° for other TMs 2, 7, 1, and 3 at the first round. Optimizing the rotations starting with TM4 at 30 ° and TM5 at 60° led to 0° preference for TM6 and other four TMs. The interhelical energy of the new structure with 30° for TM4 and 60° for TM5 was reexamined twice for conformation. Resetting TM4=30° and TM5=60° as 0, the third round let to 0 angles for all TM helixes (in pink circles) as the energetically favorable helix orientation by E-polar.

Figure 5

The two energetically favorable binding modes of SB706375 complex at rUT2R. A) the binding site of binding mode 3456 (TMs 4-5-6), B) the binding site of binding mode 1237 (TMs 1-2-7) Light color is the binding conformation before MD and dark color is the final configuration after MD for binding mode 3456 in C and binding mode 1237 in D. Hydrophilic residues in blue and hydrophobic residues in red interacting with SB706375 compound are labeled.

Figure 6

The comparison of binding energies of three SB706375 analogues in Charge Residue Model (CRM) and Neutral Residue Model (NRM). Unified cavity (UnifiedCav) in blue and Partial Delphi (PartialDel) in red are displayed with the linear equation and the r² value on the chart.

Figure 7

Two energetically favorable binding modes of SB706375 complex at hUT2R. A) and B) are the binding site of binding mode 3456 (TMs 4-5-6) and 1237 (TMs 1-2-7), respectively.

Figure 8

Comparison of the binding energies predicted for SB706375 analogues with hUT2R in the Charge Residue Model (CRM) and the Neutral Residue Model (NRM) based on the unified cavity analysis. A) Eight SB706375 analogues with binding affinities are used for binding energy analysis; B) Binding energy analysis of 8 SB analogues for both mode 1237 and mode 3456 with CRM or NRM. These results show that mode 1237 is better than mode 3456, leading to better correlation between binding affinities and binding energies.

Figure 9

Two energetically favorable binding modes of ACT058362 complex at human UT2R. The region with in the red circle displays a major difference between human and rat. A) the binding site of binding mode 3456 (TMs 3-4-5-6) and B) the binding site of binding mode 1237 (TMs 1-2-3-7). Hydrophilic residues in blue and hydrophobic residues in red surrounded by ACT-058362 compound are shown. C) Final complexes followed by molecular dynamics (MD) exhibited different preferred binding conformation of the quinoline ring at hUT2R in magenta and at rUT2R in blue. D) Light color is the binding conformation before MD and dark color is the binding E after MD.

Figure 10

The relative binding energies from Delphi method among mutant receptors of rat Uroteinsin II receptor complex with ACT058362 in binding mode 3456 and binding mode 1237. Relative binding energies before and after 10 ps quench annealing for the Charged Residue Model (CRM) (50 to 600 K) and the Neutral Residue Model (NRM) (50 to 300 K) were calculated and compared with the binding energy of wild type set to 0.