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

Abstract

For decades, the binding of prostaglandin H(2) (PGH(2)) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH(2) mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COX-downstream synthases, prostacyclin synthase (PGIS) and thromboxane A(2) synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D (1)H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution, down to within 5A, which were different than that of the U46619 bound to PGIS and U44069 (another PGH(2) mimic) bound to TXAS. These shorter distances led to further separation of the U46619 alpha and omega chains, forming a unique "rectangular" shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH(2) mimics bound to PGIS and TXAS. The studies indicated that PGH(2) can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH(2) binds to multiple target proteins even though unrelated in their protein sequences.

Figures

Figure 1

Panel I. The multiple target proteins of PGH2 synthesized from arachidonic acid (AA) through the COX pathway. The COX-1 and COX-2 enzymes convert AA to PGH2 (A), which serves as a common substrate for COX-downstream synthases including PGDS, PGES, PGFS, PGIS and TXAS, and also serves as an agonist for the TP. The chemical structures of the PGH2 mimics, U46619 (A), and U44069 (B) are also shown. Panel II. The specific ligand binding activity of the purified TP from Sf9 cells. The purified TP protein (■) in PBS was incubated with increasing amounts (0 to 60 nM) of the ligand, [3H]U46619. Unlabeled U46619 (1μM) was added to the Sf9 cells as a negative control (●) before the radio-labeled ligand was added. The results are representative data from three assays (n=3) and are shown as means ± the standard error.

Figure 2

Purified TP protein analyzed by SDS-PAGE. The FPLC-purified TP protein (approximately 5 μg) was separated by 10% SDS-PAGE, and then stained with Coomacie blue (A) or transferred to nitrocellulose membrane and analyzed by western blot using TP peptide antibody (B). The bands of the TP protein with a molecular mass of approximately 51 kD are shown with arrows.

Figure 3

1D NMR titration of U46619 bound to the purified TP. Free, unbound U46619 is shown (A). The chemical shift signals of H3 and H4 or non-chemical shift signals (H12) from the U46619 are shown, following the addition of TP protein at ratios of 67.4:1 (B), 45.7:1 (C), 23.3:1 (D), and 11.8:1 (E).

Figure 4

Intramolecular NOEs of U46619 bound to the TP protein. The new intramolecular NOEs of U46619 appeared after interaction with active TP (B) in comparison to that of its free state (A). The new NOE cross-peaks are boxed and labeled with the corresponding resonance assignments in the first and second dimensions.

Figure 5

The TOCSY spectrum showing the resonance assignments of the TP-bound U46619 in solution. The coupling network between protons on adjacent carbons is indicated.

Figure 6

Comparison of the solution structures of the PGH2 mimics determined by NMR spectroscopy. The solution structures of the U46619 (bound to the purified TP) showing the 10 superimposed NMR structures (A) and an average NMR structure in the rectangle shape (B). The U46619 is also shown alone (C) in the triangle shape, and bound to the purified PGIS [22] (D, round shape). The structure of the U44069 bound to the purified TXAS [21] (E, oval shape) is also shown.

Figure 7

Comparison of the bound and free U46619 in the TP ligand pocket. A comparison of the U46619 in bound (rectangle shape, A) and free (triangle shape, B) forms were docked into the ligand pocket of the human TP model. The human TP model was constructed by linking the NMR structures of the extracellular loops [29] to the corresponding helical structures of the TM domains generated from homology modeling using the TM domain x-ray structures of the β2 adrenergic receptor [32-33]. The region of the free form U46619 overlapped with the TM domain in an unfit position to the pocket, as indicated with the circles in the panel B.