In the alignment, the first three sequences are COX2, and the three lower are COX1. The image shows no large insertions or deletions and several highly conserved (invariant) regions in the alignment, which immediately suggests a highly conserved three-dimensional structure. The most notable differences between COX1 and COX2 are the longer N-terminal sequence of COX1 and the longer C-terminal sequence of COX2. I also marked two invariant residues on the alignment, a Tyr and an Arg. As shown below, these residues are essential in binding both substrate and many NSAID inhibitors that contain a carboxyl group. 3D structure
The left image below shows a ribbon representation of the COX1 dimer in a complex with arachidonic acid (PDB ID 1DIY). The CATH database
recognizes two domains in each monomer, the small β-hairpin domain (shown in yellow) belongs to the laminin superfamily (CATH Superfamily 184.108.40.206
). This fold is found in a large number of proteins with diverse functions. The large helical domain (red color) belongs to the heme peroxidase domain superfamily (CATH Superfamily 1.10.640.10
, orthogonal bundle). The smaller domain is located between the two enzyme subunits, presumably stabilizing the interactions between the monomers. The large domain contains the two catalytic sites. It binds the heme prosthetic group (required for both reactions, shown as a space-filling model on the image) between the cyclooxygenase and peroxidase sites. Although, to get a complex with the substrate, the heme group in this structure was replaced by Co-protoporphyrin IX, which does not support the enzymatic reaction. The helices at the bottom of the structure (residues 73-116) run parallel to the lipid membrane and attach the dimer to the lipid bilayer. The sequence alignment shows that this region is among the least conserved. As usual, clicking on the image will take you to the PDB page, where you can look closely at the structure using the graphics program. Substrate binding
The highly hydrophobic L-shaped substrate binding tunnel (shown as a space-filling model) faces the lipid membrane from which it receives the arachidonic acid (or other substrates of a similar type). The right image shows a close-up of arachidonic acid bound in the active site. I have marked some of the active site residues for orientation. As expected, the binding site is prolonged and highly hydrophobic (the substrate makes 43 hydrophobic contacts with the protein). The invariant Tyr355 and Arg120 at the entrance to the tunnel (also marked on the sequence alignment) interact with the carboxylic group of the substrate or with the polar groups of other ligands, including NSAID inhibitors. The spacious binding site explains the structural diversity of COX substrates and inhibitors. Interesting to note that the binding site of COX2 is about 25% bigger, which, as will be shown below, has implications for the design of COX2-specific inhibitors.
In the middle of the tunnel is the amino acid Ser530. This residue is the target for acetylation by aspirin, one of the most well-known inhibitors of COX enzymes. Acetylation blocks the enzymatic reaction catalyzed by COX1 and COX2, and as you may guess by now, aspirin reacts equally well with both enzymes. Although Ser530 does not participate in the enzymatic reaction, its replacement by a residue with a larger side chain inactivates the enzyme, which suggests that the correct positioning of the substrate at this location is essential for the reaction (reviewed in CA Rouzer & LJ Marnett (2020). Chem. Rev., 120, 15, 7592-7641)
. Across from Ser530 is Tyr385, a critical residue for the cyclooxygenase reaction. It donates a hydrogen atom to the heme during enzyme activation, which generates a tyrosyl radical that attacks the substrate's hydrogen atom.