title: Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry.
abstract: Aspirin and other nonsteroidal anti-inflammatory drugs target the cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the monooxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for the addition of oxygen to the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9, 2.0, and 2.4 Å, respectively. The structures reveal that (1) the acetylated Ser-530 completely blocks access to the hydrophobic groove, (2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation, and (3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove. On the basis of these structural observations, along with functional analysis of the S530T/G533V double mutant, we propose a working hypothesis for the generation of 15R-HETE by aspirin-acetylated COX-2. We also observe differential acetylation of COX-2 purified in various detergent systems and nanodiscs, indicating that detergent and lipid binding within the membrane-binding domain of the enzyme alters the rate of the acetylation reaction in vitro.
authors: Department of Structural Biology, The State University of New York at Buffalo and Hauptman-Woodward Medical Research Institute , Buffalo, New York 14203, United States.
authors: Michael J
authors: MJ
authors: Lucido
authors: Department of Structural Biology, The State University of New York at Buffalo and Hauptman-Woodward Medical Research Institute , Buffalo, New York 14203, United States.
authors: Benjamin J
authors: BJ
authors: Orlando
authors: Department of Structural Biology, The State University of New York at Buffalo and Hauptman-Woodward Medical Research Institute , Buffalo, New York 14203, United States.
authors: Alex J
authors: AJ
authors: Vecchio
authors: Department of Structural Biology, The State University of New York at Buffalo and Hauptman-Woodward Medical Research Institute , Buffalo, New York 14203, United States.
authors: Michael G
authors: MG
authors: Malkowski
mesh_terms: D000107:Acetylation / Q000187:drug effects*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry* / Q000494:pharmacology; D001241:Aspirin / Q000737:chemistry* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D062105:Molecular Docking Simulation; D011487:Protein Conformation
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D051546:Cyclooxygenase 2; D001241:Aspirin