Abstract
The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 Å, respectively, and R513H muCOX-2 in complex with AA to 2.45 Å resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.
Highlights
20736 JOURNAL OF BIOLOGICAL CHEMISTRY generated from the fatty acid substrate arachidonic acid (AA), represents the committed step in the biosynthesis of prostaglandins and thromboxanes
AA is oriented in the cyclooxygenase channel with the carboxylate group of the substrate located near Arg-120 and Tyr-355 at the opening of the channel and the -end of the substrate bound in a hydrophobic groove above Ser-530 [9, 10]
We report here studies designed to investigate the structural basis of endocannabinoid substrate binding to COX-2
Summary
Materials—The COX-2 substrates AA (5Z,8Z,11Z,14Zeicosatetraenoic acid), 2-AG (1,3-dihydroxy-2-propanyl 5Z,8Z,11Z,14Z-eicosatetraenoic acid), 1-AG (2,3-dihydroxypropyl 5Z,8Z,11Z,14Z-eicosatetraenoic acid), 2-AGe (2-[(5Z,8Z,11Z,14Z)-5,8,11,14-icosatetraen-1-yloxy]-1,3-propanediol), N-arachidonoyl glycine (N-[1-oxo-5Z,8Z,11Z,14Zeicosatetraenyl]-glycine, AEA (N-(2-hydroxyethyl)-5Z,8Z, 11Z,14Z-eicosatetraenamide, and S-1 methanandamide (N-(2hydroxy-1S-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) were purchased from Cayman Chemical Company (Ann Arbor, MI). Following centrifugation at 40,000 rpm at 4 °C for 1 h, the supernatant was loaded on a 5-ml HiTrap Chelating HP column and washed with buffer A (50 mM Tris, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, and 0.5% (w/v) decyl maltoside). The column was washed with buffer B (buffer A containing a final concentration of 60 mM imidazole), protein eluted with buffer C (buffer A containing 200 mM imidazole), followed by pooling and dialysis overnight at 4 °C against 50 mM Tris, pH 8.0, 300 mM NaCl, and 0.53% (w/v) OG for utilization in functional studies. The trypsinized His N580A muCOX-2 was subjected to size exclusion chromatography utilizing a HiPrep 16/60 Sephacryl S-300 HR column equilibrated in 25 mM Tris, pH 8.0, 150 mM NaCl, and 0.53% (w/v) OG.
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