Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique

Understanding the docking mechanism of the common substrate, prostaglandin H2 (PGH2), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH2 to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH2 analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A2 synthase (TXAS). The dynamic binding was clearly observed by 1D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D 1H NMR experiments using transferred NOEs. Through the assignments for the 2D 1H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH2 mimic of the TXAS in solution, and to imply that PGH2 undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH2 to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH2 bound to other COX-downstream synthases.