STRUCTURAL BIOLOGY OF PROSTAGLANDIN BIOSYNTHESIS

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Prostaglandin H synthase (PGHS) is a an integral membrane protein which converts arachidonic acid, an essential fatty acid, into the initial enzymatic products, PGG2 and PGH2 by means of a free radical mechanism. Prostaglandins initiate numerous cell and tissue responses including vasodilation, vaso-constriction, blood clotting, cell proliferation and inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostanoid biosynthesis by targeting the cyclooxygenase activity of PGHS, are used to treat certain symptoms of inflammatory and cardiovascular diseases. The prostanoids are also involved in pathophysiology of a number of diseases including atherosclerosis, rheumatoid arthritis, and cancer; in the latter, aspirin is now a proven anticancer prophylaxis. Two isoforms of PGHS have been discovered: PGHS-1 is involved in homeostatic or "house- keeping" prostaglandin biosynthesis while PGHS-2 induced by cytokines during inflammatory events. We are continuing our efforts to determine the X-ray crystal structure of PGHS-1 to the highest resolution possible. We have solved the structure of PGHS-1 to 3.1 A resolution, have built an atomic model of the enzyme, and have characterized PGHS-1 complexes a=with four NSAIDs: bromoaspirin, flurbiprofen, iodosuprofen and iodoindomethacin. We are beginning to collect high resolution data (2.8 A) at the synchrotron radiation facilities which will require further technological development such as the flash-freezing of membrane protein crystals. We will continue our study on the nature of enzyme-drug interactions and structure-function relationships, with particular focus on the mechanisms of substrate and NSAID interactions as well as the mode of targeting the ER membrane. This would involve analyzing ligand-induced conformational changes in PGHS-1, and characterizing ligand complexes of PGHS-1 and PGHS-2 by means of a complementary series of enzymological, EPR/ENDOR spectroscopic and X-ray diffraction experiments. With the discovery of an inducible from of PGHS, questions arise about the functional differences between PGHS-1 and PGHS-2. We have predicted the structure of PGHS-2, using the known sequences and the PGHS-1 as a model, and are analyzing the structure-function relationships in this new isozyme. Crystallization trial for mouse PGHS-2 are underway.