Identification of Residues Important for Ligand Binding of Thromboxane A2 Receptor in the Second Extracellular Loop Using the NMR Experiment-guided Mutagenesis Approach

Abstract

The second extracellular loop (eLP2) of the thromboxane A 2 receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional 1 H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001 Thromboxane A 2 (TXA 2 ) 1 is a potent platelet aggregatory and vasoconstrictive mediator (2). The function of TXA 2 is mediated by specific cell surface receptor, thromboxane A 2 receptor (TP) (3). The understanding of the structure and function of TP receptor can greatly explain how the ligand binds to its receptor and initiates the following cell signaling.TP receptor was first purified from platelet in 1989, and the cDNA of TP receptor was cloned from placenta in (4, 5). Other human prostanoid receptor cDNAs have also been cloned by homology screening. All of the prostanoid receptors belong to the G-protein-coupled receptor family that share a basic seven transmembrane segments and couple to different signal transduction systems to play diverse physiological and pathological roles (6 -14). TXA 2 binds to TP receptor and triggers an increase of intracellular calcium. There were two TP receptor isoforms with different C-terminal tails, resulting from alternative splicing that the last 15 amino acids of the C terminus were replaced by 79 amino acids (15, 16). The two TP receptor isoforms coupled to the same signal transduction, but endothelium expressed only the spliced form and placenta expressed both types of the TP receptors (15-17).Based on the sequence alignment, the second extracellular loop (eLP2) and the third and seventh transmembrane domains of the prostanoid receptors are highly conserved and are proposed to be involved in ligand binding (18). in the eLP2 of the EP3 receptor have been reported as an essential determinant of ligand selectivity (19). These results suggest that the extracellular domains of other prostanoid receptors are involved in the initial specific ligand interaction. The residues responsible for specific ligand recognition within eLP2 of the TP receptor have not been thoroughly examined. The mutations based on alignment only are controversial and will need structural information to support. The structures of the transmembrane domains of prostanoid receptors may be similar, but the specific recognition sites on extracellular domains will be different because the ligand structures are different. Thus, structural characterization of the extracellular functional domains of prostanoid receptors could help in understanding the specificities of ligand binding. In our current study, the structure of the highly conserved eLP2 has been characterized by high resolution NMR using a synthetic eLP2 peptide with constrained loop ends (1). To identify which residues make up the ligand recognition site of the receptor, SQ29,548 was added to the peptide to determine the interaction using high resolution two-dimensional 1 H NMR technique...

Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: Implication of the G protein-coupling pocket

“…Based on the TM domain model of the human TP receptor that was previously created by homology modeling using the X-ray structure of bovine rhodopsin as a template [29][30][31][32], the distance between the iLP2 termini, connected to the third and fourth TM domains is about 12.9 Å and the distance between the iLP3 termini connected to the fifth and sixth TM domains is about 12.4 Å ( Figure 1A). Two peptides corresponding to the TP iLP2 and TP iLP3 regions ( Figure 1B), which contained additional homocysteine residues at the N-and C-termini, were synthesized and constrained to a similar distance at the termini by a disulfide bond formation using a the fluorenyl-methoxy-carbonyl-polyamide solid phase method ( Figure 1C) [27,[29][30][31][32][33][34][35][36]. After synthesis, the peptides were purified to homogeneity by reverse phase HPLC using a C4 column, and the correct molecular weights were confirmed by mass spectrometry analysis [37].…”

“…1H 2D TOCSY, NOESY and DQF-COSY spectra were used to measure the constrained peptides. 1H NMR assignments were accomplished using the standard sequential assignment technique described [26,27,[29][30][31][32][33][34][35][36]. The assignment procedures involved in the identification of spin systems using the TOCSY spectra, and the sequential assignments using NOESY spectra were completed by manual assignment.…”

Characterization of the prostaglandin H2 mimic: Binding to the purified human thromboxane A2 receptor in solution

“…The solution NMR structures of the TP extra- [29] and intra- [30] cellular loop segments were used to link the TM segments together to form a 3D TP model by using the Insight II protein modeling software. The PGH 2 mimics were docked into the ligand pocket that was previously predicted by NMR spectroscopic and mutagenesis studies [29,31], and by taking into account the ligand pocket in the β2 adrenergic receptor [32][33]. The docking study was performed by a 3D-Quantitative Structure Activity [34] using SYBYL Surflex-Dock software (Tripos, St. Louis, MO).…”

“…The TP extracellular loop structure was constructed by linking the NMR structures of the three loops to the corresponding TM domains and then optimized by energy minimization as described [29]. The TM ligand-binding site of the TP was localized by using previous mutagenesis information [31], which includes the interaction of the TP extracellular loop with its ligand, and the ligand pocket in the crystal structure of the β2 adrenergic receptor [32][33]. The NMR structure of the TP-bound U46619 conformation ( Figure 6B, rectangle shape) could fit into the putative ligand pocket very well ( Figure 7A).…”

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A2 Receptor in Full Size for Structural Studies

“…Recently, through a combination of the NMR spectroscopy technique, site-directed mutagenesis, peptide mimicking, and molecular modeling (21)(22)(23)(24)(25)(26) we have revealed the ligand-recognition site in the extracellular domains and identified that the second extracellular loop (22,25,26) plays a critical role in forming the ligand-recognition pocket (22,(24)(25)(26). However, it remains critical to understand the detailed molecular mechanisms for the ligand recognition, binding, and signaling activation of the native receptor.…”