Crystallization of recombinant cyclo-oxygenase-2

• Published in: Journal of crystal growth 1999-01, Vol.196 (2), p.350-355
• Main Authors: Stevens, Anna M., Pawlitz, Jennifer L., Kurumbail, Ravi G., Gierse, James K., Moreland, Kirby T., Stegeman, Roderick A., Loduca, Jina Y., Stallings, William C.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Crystallization; Cyclo-oxygenase; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Membrane protein; NSAID (nonsteroidal anti-inflammatory drug); Prostaglandin H 2 synthase; Proteins
• ISSN: 0022-0248; 1873-5002
• DOI: 10.1016/S0022-0248(98)00847-1

The integral membrane protein, prostaglandin H 2 synthase, or cyclo-oxygenase (COX), catalyses the first step in the conversion of arachidonic acid to prostaglandins (PGs) and is the target of nonsteroidal anti-inflammatory drugs (NSAIDs). Two isoforms are known. The constitutive enzyme, COX-1, is present in most tissues and is responsible for the physiological production of PGs. The isoform responsible for the elevated production of PGs during inflammation is COX-2 which is induced specifically at inflammatory sites. Three-dimensional structures of inhibitor complexes of COX-2, and of site variants of COX-2 which mimic COX-1, provide insight into the structural basis for selective inhibition of COX-2. Additionally, structures of COX-2 mutants and complexes with the substrate can provide a clearer understanding of the catalytic mechanism of the reaction. A crystallization protocol has been developed for COX-2 which reproducibly yields diffraction quality crystals. Polyethyleneglycol 550 monomethylether (MMP550) and MgCl 2 were systematically varied and used in conjunction with the detergent β- D-octylglucopyranoside ( β-OG). As a result of many crystallization trials, we determined that the initial β-OG concentration should be held constant, allowing the salt concentration to modulate the critical micelle concentration (CMC) of the detergent. Over 25 crystal structures have been solved using crystals generated from this system. Most crystals belong to the space group P2 12 12, with lattice constants of a=180, b=134, c=120 Å in a pseudo body-centered lattice.