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

摘要: Abstract For decades, the binding of prostaglandin H2 (PGH2) to multiple target proteins unrelated protein structures which mediate diverse biological functions has remained a real mystery in field eicosanoid biology. Here, we report that structure PGH2 mimic, U46619, bound purified human TP, was determined and compared with its conformation COX-downstream synthases, prostacyclin synthase (PGIS) thromboxane A2 (TXAS). Active TP protein, glycosylated full length, expressed Sf-9 cells using baculovirus (BV) expression system then near homogeneity. The U46619 receptor nonionic detergent-mimicked lipid environment characterized by high-resolution NMR spectroscopy. conformational change upon active evidenced significant perturbation chemical shifts protons at H3 H4 concentration-dependent manner. detailed changes 3D from free form TP-bound were further solved 2D 1H experiments transferred NOE (trNOE) technique. distances between H11 H18, H19, H15 H19 shorter following their solution, down within 5 A, different than PGIS U44069 (another mimic) TXAS. These led separation α ω chains, forming unique “rectangular” shape. This enabled molecule fit into ligand-binding site pocket model, homology modeling used for transmembrane (TM) domain, extramembrane loops. proton perturbations conformations mimics studies indicated can adopt solution satisfy specific shapes pockets enzymes. results also provided sufficient information speculating molecular basis how binds even though sequences.