NMR structure of the thromboxane A₂ receptor ligand recognition pocket

Abstract

To overcome the difficulty of characterizing the structures of the extracellular loops (eLPs) of G protein-coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate a three-dimensional structural model for a GPCR, the thromboxane A 2 receptor. This three-dimensional structure was completed by the assembly of the NMR structures of the computation-guided constrained peptides that mimicked the extracellular loops and connected to the conserved seven transmembrane domains. The NMR structure-based model reveals the structural features of the eLPs, in which the second extracellular loop (eLP 2 ) and the disulfide bond between the first extracellular loop (eLP 1 ) and eLP 2 play a major role in forming the ligand recognition pocket. The eLP 2 conformation is dynamic and regulated by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition. The reduced form of the thromboxane A 2 receptor experienced a decrease in ligand binding activity due to the rearrangement of the eLP 2 conformation. The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP 2 conformation. This molecular mechanism of ligand recognition is the first that may be applied to other prostanoid receptors and other GPCRs.

TxA 2 Receptor‐Based Vaccination: A Novel Potential Therapeutic Approach to Limit Thrombosis

“…To achieve this, the authors immunized the mice through a peptide‐based vaccination approach using the TPR C‐EL2 (C‐terminus of the second extracellular loop) peptide. Previously, these authors and other groups mapped the TPR ligand binding domain and found that it lies between C 183 and D 193 regions of C‐EL2 9, 10. Taking these studies further, authors have previously characterized C‐EL2Ab (antibody against C‐EL2 domain) and showed that the antibody selectively blocks TPR‐mediated platelet aggregation 11.…”

Investigation of a Thromboxane A 2 Receptor–Based Vaccine for Managing Thrombogenesis

“…One apparent reason for this failure is because these agents were empirically designed on the basis of the complex structures of prostaglandin H 2 and/or TXA 2 , with little information on the actual TPR binding domains. In this connection, research efforts11, 30, 39, 40, 41 by us and others to map the TPR ligand‐binding domain revealed the following: (1) the ligand‐binding domain resides in the C‐terminus of the second extracellular loop (C‐EL2; C 183 ‐D 193 ) of the receptor protein; (2) this extracellular segment contains ligand–amino acid coordination sites; and (3) an antibody raised against this sequence (ie, abbreviated C‐EL2Ab) inhibits TPR ligand binding and platelet aggregation and protects from thrombogenesis without any apparent bleeding diathesis, making it the first functional antibody against platelet TPRs. Based on these considerations, we sought to further assess the contributions of the C‐EL2 domain to in vivo TPR‐dependent platelet activation (eg, hemostasis/bleeding time) and the genesis of thrombosis, by using a vaccine‐based approach using the cognate TPR C‐EL2 peptide as an immunogen.…”

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

“…A structural model of the human TP receptor, based on the extracellular loops' structures determined by NMR spectroscopy and the conserved TM domains generated from homology modeling using bovine rhodopsin, has been characterized and reported [29]. Also, the crystal structure of the human β2 adrenergic receptor, a member of the G protein-coupled receptor (GPCR) family, was very recently reported [32][33].…”

“…A TP seven-TM domain model ( Figure 7, green colors) was generated by homology modeling using the x-ray structure of the TM domains from the β2 adrenergic receptor as templates. 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 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).…”