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.

Figure 1. Aspirin-acetylation and Salicylate Binding within the Cyclooxygenase Channel of huCOX-2

(A) Stereo view of the aspirin-acetylated Ser-530 side chain at the apex of the cyclooxygenase channel of monomer B in the huCOX-2:ASA crystal structure. The F_O-F_C simulated annealing omit map electron density (blue), contoured at 3.0σ, is shown for the acetylated Ser-530 side chain (pink carbon atoms and red oxygen atoms). (B) Stereo view depicting hydrogen bond (red dashes) and hydrophobic contact (black dashes) distances between the side chains of Val-344, Tyr-348, Val-349, Tyr-385, and acetylated Ser-530. (C) Stereo view of salicylate bound within the cyclooxygenase channel of monomer A in the huCOX-2:SAL crystal structure. The F_O-F_C simulated annealing omit map electron density (blue), contoured at 3.0σ, is shown for salicylate (yellow carbon atoms and red oxygen atoms). Residues lining the cyclooxygenase channel are labeled accordingly, with carbon, nitrogen, and oxygen atoms colored green, blue, and red, respectively.

Figure 2. Comparison of Acetylated and Salicylate-bound huCOX-2 with Bromine-acetylated ovCOX-1

(A) Stereo view of the superposition of the acetylated Ser-530 side chain from monomer B of huCOX-2:ASA with the bromine-acetylated Ser-530 side chain from ovCOX-1 (PDB ID 1PTH; ). (B) Stereo view of the superposition of salicylate (yellow carbon atoms and red oxygen atoms) from monomer B of huCOX-2:SAL with salicylate bound in the cyclooxygenase channel of ovCOX-1 from 1PTH. The salicylate-leaving group from 1PTH is colored with orange carbon atoms and red oxygen atoms. Residues lining the cyclooxygenase channel of COX-2 are labeled accordingly, with carbon atoms colored green, while those from COX-1 are colored sky blue. Nitrogen and oxygen atoms in both structures are colored dark blue and red, respectively.

Figure 3. AA access to the hydrophobic groove in S530T muCOX-2

(A) Schematic of the cyclooxygenase channel from the S530T muCOX-2 crystal structure superimposed onto the wild type muCOX-2 crystal structure complexed with AA (pdb 3HS5; ). The ω-end of AA (yellow) would have full access to the hydrophobic groove in the S530T mutant. (B) Superposition of the aspirin-acetylated huCOX-2 crystal structure onto the view in A. Residues lining the cyclooxygenase channel are labeled accordingly, with carbon, nitrogen, and oxygen atoms colored green, blue, and red, respectively.

Figure 4. Inhibition of huCOX-2 in nanodiscs and various detergent systems

Inhibition assays were performed as detailed in Experimental Methods to assess time-dependent and time-independent inhibition of huCOX-2 in CHAPS, Tween-20, C₁₀E₆, βOG, and nanodiscs. (A) Time-independent inhibition was assessed using (R/S)-IBP. To assess time-dependent inhibition, a time course was performed and residual cyclooxygenase activity was measured at defined time points of 0.5, 5, 12.5, 20, and 30 min using (B) (R/S)-FBP and (C) and celecoxib (CBX).

Figure 5. Differential acetylation of huCOX-2 in nanodiscs and various detergent systems

(A) Aspirin inhibition of huCOX-2 in CHAPS (purple), Tween-20 (black), C₁₀E₆ (red), βOG (green), and nanodiscs (blue) measured over time using an oxygen electrode. Values represent the average of triplicate measurements ± S.E.M. (B) SDS-PAGE and (C) radiographic analysis of the acetylation of huCOX-2 by [acetyl-¹⁴C] ASA. The percentages represent the intensity of the radioactive band relative to huCOX-2 prepared in βOG. Lane 1, nanodisc-reconstituted huCOX-2; Lane 2, huCOX-2 in βOG; Lane 3, huCOX-2 in CHAPS; Lane 4, huCOX-2 in C₁₀E₆; Lane 5, huCOX-2 in Tween-20. Note, the lower molecular weight band in Lane 1 corresponds to the MSP of the nanodisc. MW, molecular weight marker. (D) TLC analysis of the products generated from [1-¹⁴C] AA by huCOX-2 in the absence (−) or presence (+) of aspirin. PGs, prostaglandin products; HHT, 12-hydroxyheptadecatrienoic acid; HETE, 11- and 15-hydroxyeicosatetraenoic acids. HHT is produced via nonenzymatic decomposition of PGs.

Figure 6. Model for 15R-HETE generation by acetylated huCOX-2

A model of AA (yellow) bound within the cyclooxygenase channel of aspirin-acetylated huCOX-2. The acetylated Ser-530 side chain (pink carbon atoms and red oxygen atoms) is proposed to adopt an alternate conformation, subsequently providing the ω-end of AA access to the hydrophobic groove to achieve a productive binding pose leading to the generation of 15R-HETE. In conjunction, the side chain of Leu-531 (blue) would also adopt an alternate conformation (orange) to accommodate the moving acetylated side chain and the binding of AA within the cyclooxygenase channel. Residues lining the cyclooxygenase channel are labeled accordingly, with carbon, nitrogen, and oxygen atoms colored green, blue, and red, respectively.