A strategy using NMR peptide structures of thromboxane A2 receptor as templates to construct ligand-recognition pocket of prostacyclin receptor

Abstract

Background: Prostacyclin receptor (IP) and thromboxane A2 receptor (TP) belong to rhodopsin-type G protein-coupling receptors and respectively bind to prostacyclin and thromboxane A2 derived from arachidonic acid. Recently, we have determined the extracellular loop (eLP) structures of the human TP receptor by 2-D 1H NMR spectroscopy using constrained peptides mimicking the individual eLP segments. The studies have identified the segment along with several residues in the eLP domains important to ligand recognition, as well as proposed a ligand recognition pocket for the TP receptor.

Results: The IP receptor shares a similar primary structure in the eLPs with those of the TP receptor. Forty percent residues in the second eLPs of the receptors are identical, which is the major region involved in forming the ligand recognition pocket in the TP receptor. Based on the high homology score, the eLP domains of the IP receptor were constructed by the homology modeling approach using the NMR structures of the TP eLPs as templates, and then configured to the seven transmembrane (TM) domains model constructed using the crystal structure of the bovine rhodopsin as a template. A NMR structure of iloprost was docked into the modeled IP ligand recognition pocket. After dynamic studies, the segments and residues involved in the IP ligand recognition were proposed. A key residue, Arg173 involved in the ligand recognition for the IP receptor, as predicted from the modeling, was confirmed by site-directed mutagenesis.

Conclusion: A 3-D model of the human IP receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin TM domains and the NMR structures of the synthetic constrained peptides of the eLP domains of the TP receptor as templates. This strategy can be applied to molecular modeling and the prediction of ligand recognition pockets for other prostanoid receptors.

Figures

Figure 1

Homology modeling of the seven TM domains of human IP receptor. 3-D backbone structure of the seven TM domains of the human TP receptor were created by homology modeling using the crystal structural backbones of the TM domains of the bovine rhodopsin as templates.

Figure 2

Sequence alignment of the eLPs between the human IP and TP receptors. The sequences of the putative eLP domains of the human IP (45) and TP (3) receptors were aligned by a sequence alignment program in Insight II software package and manual adjustment. The identical and highly similar residues between the TP and IP are shaded. The residues in the eLP2 regions previously identified important to the ligand binding for the TP receptor (10) and their corresponding residues in the IP receptor are underlined.

Figure 3

The predicted backbone structures of the three eLP domains of the human IP receptor. The 3-D structural models of the eLPs were constructed by homology modeling with the NMR structures of the eLP2 (48), the eLP3 (49) and the eLP1 (unpublished data) of the human TP receptor as templates using the molecular modeling package of Insight II and Discover software packages. The conformation of the eLP2 is placed in a position with respect to the formation of a disulfide bond between the Cys92 in the eLP1 and Cys170 in the eLP2.

Figure 4

Configuration of the modeled 3-D backbone structures of the three IP eLP domains (Figure 4) onto the working model of the seven TM domains of the IP receptor. Before (A) and after (B) the connections of the eLP structures to the seven TMs through chemical bonds are displayed for their comparison.

Figure 5

Ligand docking with the eLP domains of the IP receptor. The four residues including Gln162, Leu172, Arg173 and Met174 in the IP eLP2 (eLP1, bleu color; eLP2, red color and eLP3, yellow color) in contact with iloprost were predicted by the sequence alignment (Figure 1) using the identified four residues (Val176, Leu185, Thr186 and Leu187) in the TP eLP2 contacted with SQ29,548 (10) as a template. 3-D NMR structure of the IP receptor agonist, iloprost (42) was docked into the putative ligand recognition pocket formed by the three eLPs with respect to the contacts with Leu172, Arg1173 and Met174 in the opening of the pocket. In addition, the two residues, Ala177 and Gln178 involved in contacts with iloprost predicted by NMR spectroscopic studies (42) were also used as constraints for the iloprost docking to the recognition pocket. The configuration of the model was minimized using 1,000-step energy minimization after the iloprost was docked into the pocket. The TM domains of the IP receptor are showed with green colors.

Figure 6

Analysis for the mutation of Arg173 to Ala residue of the recombinant human IP receptor. A). Western blot analysis. Fifty micrograms of COS-7 cells transfected with wild-type (WT) or a mutant IP receptor (R173A) cDNA was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with rabbit anti-IP peptide antibody. (B) The ligand binding activities of wild-type and mutant TP receptor. 300 μg of the protein prepared from the COS-7 cells transfected with cDNA of the wild-type (WT) or the R173A mutant was incubated with 4 nM [3H]-iloprost (30,000 cpm) in the absence or presence of unlabeled iloprost (1 μM) in a reaction volume of 100 μl. After 1 h incubation, the reaction was stopped and the binding activity of the recombinant IP receptor was measured as described in the methods. The binding activity of wild-type receptor was considered as 100% (2,000 cpm).

Figure 7

Analysis of the mutation of Arg173 to Thr residue and Ser168 to Thr residue of the recombinant IP receptors. A). Western blot. B). Ligand binding activity. The methods are described in the Figure 6.

Figure 8

Kinetic properties of [3H] iloprost binding to the recombinant IP receptors expressed in COS-7 cells. The cell membrane protein prepared from COS-7 cells that transiently expressed the wild-type (squares), R173A mutant (circles) or R173T mutant (triangles) of the IP receptor was incubated with the increasing concentration of the [3H] iloprost.