Understanding the docking mechanism of the common substrate, prostaglandin H 2 (PGH 2 ), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH 2 to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH 2 analogue, bound to one of the COX-downstream synthases—an engineered thromboxane A 2 synthase (TXAS). The dynamic binding was clearly observed by 1 D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D 1 H NMR experiments using transferred NOEs. Through the assignments for the 2D 1 H 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 PGH 2 mimic of the TXAS in solution, and to imply that PGH 2 undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH 2 to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH 2 bound to other COX-downstream synthases.