Crystallization of recombinant cyclo-oxygenase-2

The integral membrane protein, prostaglandin H-2 synthase, or cyclo-oxygena se (COX), catalyses the first step in the conversion of arachidonic acid to prostaglandins (PGs) and is the target of nonsteroidal anti-inflammatory d rugs (NSAIDs). Two isoforms are known. The constitutive enzyme, COX-1, is p resent in most tissues and is responsible for the physiological production of PGs. The isoform responsible for the elevated production of PGs during i nflammation is COX-2 which is induced specifically at inflammatory sites. T hree-dimensional structures of inhibitor complexes of COX-2, and of site va riants of COX-2 which mimic COX-1, provide insight into the structural basi s for selective inhibition of COX-2. Additionally, structures of COX-2 muta nts and complexes with the substrate can provide a clearer understanding of the catalytic mechanism of the reaction. A crystallization protocol has be en developed for COX-2 which reproducibly yields diffraction quality crysta ls. Polyethyleneglycol 550 monomethylether (MMP550) and MgCl2 were systemat ically varied and used in conjunction with the detergent beta-D-octylglucop yranoside (beta-OG). As a result of many crystallization trials, we determi ned that the initial beta-OG concentration should be held constant, allowin g the salt concentration to modulate the critical micelle concentration (CM C) of the detergent. Over 25 crystal structures have been solved using crys tals generated from this system. Most crystals belong to the space group P2 (1)2(1)2, with lattice constants of a = 180, b = 134, c = 120 Angstrom in a pseudo body-centered lattice. (C) 1999 Elsevier Science B.V. All rights re served.