COX-1/Prostaglandin H2 Synthase (PDB ID: 1CQE) from Ovis aries
Created by: Hannah Ashe
COX-1/Prostaglandin H2 Synthase (PDB ID: 1CQE) is an enzyme in the cyclooxygenase subgroup of monotopic membrane proteins found in Ovis aries. It is primarily located in the endoplasmic reticulum. The main function of the protein is to synthesize prostaglandin H2 (PGH2) from arachidonic acid using an intermediate, prostaglandin G2 (PGG2) which is involved in maintaining tissue homeostasis and in cell-cell signaling. This is the first committed step of prostaglandin synthesis (1). Prostaglandins are lipids involved in pathogenic pathways such as the inflammatory response and platelet aggregation and have many derivatives of pathophysiological importance like thromboxane and prostacyclin (2). Because of its integral role in prostaglandin biosynthesis, COX-1/Prostaglandin H2 Synthase (COX-1/PGHS) is a target for non-steroidal anti-inflammatory drugs (NSAIDs). The inhibition of COX-1/PGHS with NSAIDs is of particular interest to scientists because it has been shown to reduce the incidence of cardiovascular disease (1,2).
COX-1/Prostaglandin H2 Synthase from Ovis aries was obtained from sheep seminal vesicles and purified. To prepare for crystallization, a 10 mg/ml sample was dialyzed in 20 mM sodium phosphate at pH 6.7, 100-200 mM NaCl, 0.6% (w/ v) β-octyl glucopyranoside, and 0.1 mM flurbiprofen was then diluted to a 4% solution using polyethylene glycol 4000. The protein was crystallized through hanging-drop vapor diffusion using the aforementioned buffer concentrated at 4X and 4-8% polyethylene glycol 4000. The structure of COX-1/Prostaglandin H2 Synthase was determined using X-ray crystallography at a resolution of 3.5 Å (1).
The molecular weight of COX-1/Prostaglandin H2 Synthase is 133020.99 Da, and the isoelectric point is 6.89 determined by the Expasy database (3). COX-1/PGHS is a homodimer of two monomers. The two subunits are structurally and functionally identical. Each asymmetric subunit has three structural domains: the epidermal growth factor-like (EGF-like) domain (residues 34-72), the membrane binding domain or MBD (residues 73-116), and the globular catalytic domain (residues 117-587) (1,2). COX-1/PGHS was crystallized for residues 33-586 (1), so all three subunits were present in the crystallized structure. However, the two-fold symmetry axis was unable to be crystalized (1). Four ligands were present in the crystallized structure: heme prosthetic group, N-acetyl-D-glucosamine, B-octylglucoside, and flurbiprofen. N-acetyl-D-glucosamine and B-octylglucoside were present to induce crystallization while the heme group and flurbiprofen serve functional roles (4).
The primary structure of COX-1/Prostaglandin H2 Synthase contains a total 1160 residues with 580 residues composing each subunit (4). The amino acid sequence is composed of both hydrophobic and hydrophilic amino acids (5). The secondary structure is 43% α-helix (31 helices, 250 residues per subunit), 9% β-sheet (31 strands, 53 residues per subunit), and the rest is random coils (4). The α-helices in the protein are amphipathic, matching the mixed polarities of the amino acid sequence. This is integral for the protein as it allows for the protein to be inserted in the lipid bilayer, so hydrophobic surfaces face outward and hydrophilic surfaces face inward (1). The hydrogen bonding between the carbonyl oxygens and amide hydrogens also provide structural stabilization for the protein.
The tertiary structure of COX-1/Prostaglandin H2 Synthase contains three distinct folding units. The first is the epidermal growth factor-like (EGF-like) domain that is small and compact, held together by three intra-domain disulphide linkages between cysteine residues (1). This domain is involved with the folding of the protein (5). It also contains a fourth disulfide linkage (Cys-37 – Cys-159) that connects this domain to the main body of the enzyme (1). The second domain is the membrane binding domain (MBD) consisting of a right-handed spiral of α-helices (1). Here is where the amphipathic α-helices, with hydrophobic residues sticking out, anchors the protein to the lipid bilayer. An additional α-helix associates with the main body of the enzyme to form the third folding unit. The third domain is the globular catalytic domain which includes the cyclooxygenase active site and the peroxidase active site (2). This domain can be further subdivided into two lobes, both made up of α-helices. The domain also contains a hydrophobic channel which is the location for substrate binding for the cyclooxygenase active site(1), and hydrophilic water channels that play a structural and/or catalytic role. The quaternary structure of the COX-1/PGHS is the association between the two subunits. As previously mentioned, the protein has two identical subunits that associate at a dimer interface primarily formed by the EGF domain (5).
The biosynthesis of prostaglandin H2 from arachidonic acid occurs in the globular catalytic domain of COX-1/Prostaglandin H2 Synthase and occurs in two main stages. The pathway begins with the conversion of arachidonic acid to the intermediate PGG2 in the cyclooxygenase active site. The cyclooxygenase active site is a long, narrow, hydrophobic channel with Tyr-385 at the apex, proximal to a heme group. The substrate, arachidonic acid, lies between Tyr-385 and Arg-120. Arg-120 is one of two polar residues in the channel, so it interacts with the carboxylate of arachidonic acid. The reaction begins with the removal of the pro-S hydrogen of arachidonic acid to create a radical on C-13 and the addition of molecular oxygen at C-11. The initial removal of the pro-S hydrogen is due to a tyrosyl radical of Tyr-385, which forms by its interaction with the ferryloxy porphyrin cation radical (the heme group) created by the peroxidase active site. This creates a peroxyl radical that initiates cyclization forming 9,11-endoperoxide. An addition molecule of oxygen then enters and forms another peroxyl radical; a hydrogen atom gets donated back to the substrate to form PGG2. This stage of prostaglandin synthesis is targeted by NSAIDs, specifically flurbiprofen. NSAIDs acetylate Ser-530 which is located near Tyr-385 at the channel opening. The acetylation blocks access to the active site and therefore inhibits the activity of the enzyme (1).
The second stage of prostaglandin biosynthesis by COX-1/Prostaglandin H2 Synthase occurs in the peroxidase active site of the globular catalytic domain. Here, PGG2 produced by the cyclooxygenase step gets reduced to PGH2 by a two-electron transfer reaction. In the process, a ferryloxy porphyrin cation radical is formed – the radical used to initiate the cyclooxygenase reaction. The peroxidase active site is located in a cleft between the two lobes of the domain and contains a heme prosthetic group. The heme is coordinated with His-388. His-207 and Gln-203 partially enclose the active site, although much of the active site is exposed to accommodate the size of PGG2 (1). His-207 and Gln-203 are both important for catalytic activity. His-207 is important in de/protonation, and Gln-203 does not yet have a resolved function, but when either are mutated, the peroxidase activity is reduced or eliminated (5).
In order to find comparison proteins for COX-1, two programs were used. The first program was PSI-BLAST. The purpose of PSI-BLAST is to find proteins with similar primary structures by searching protein databases using definitional, statistical, and algorithmic refinements. PSI-BLAST assigns an E value to each similar primary structure, referred to as subjects. The smaller the E value, the closer the more similar the subject is to the query protein. E values less than 0.05 are considered to be significant. The other program used was the Dali Server. The Dali Server compares tertiary structures of proteins and calculates the differences in intramolecular distances using a sums-of-pairs method and listed as Z scores. A score above 2 is considered significant and means the comparison protein has similar folds to the protein of interest.
An important comparison to make is between COX-1/Prostaglandin H2 Synthase and its isozyme, COX-2/Prostaglandin H2 Synthase 2(PDB ID: 3QH0). COX-2 is found in Mus musculus in the endoplasmic reticulum. COX-1 and COX-2 share a 62.94% similarity in their primary structures with an E value of 0.0 (6). COX-2 has 1220 amino acid residues. Their secondary structures are similar as well, with COX-2 being 42% α-helix (32 helices, 261 residues), 8% β-sheet (30 strands, 50 residues), and the rest random coiling (4). Using Dali server, they share a 65% similarity in their tertiary structurewith a Z score of 58.6 (7). COX-2 has the same three domains: EGF-like domain, membrane binding domain, and globular catalytic domain. COX-2, like COX-1, is a homodimer of two asymmetric monomeric units.
Functionally, COX-1 and COX-2 are very similar. The primary function of both enzymes is to convert arachidonic acid to prostaglandin H2. Like COX-1, COX-2 has a cyclooxygenase active site that converts arachidonic acid to the intermediate PGG2 and a peroxidase active site that converts PGG2 to PGHS and activates the cyclooxygenase site through a ferrooxy porphyrin cation radical. The positions of the important amino acid residues are comparable as well. The apex of the hydrophobic channel of the cyclooxygenase active site has Tyr-385 and Ser-530. Substrates in the cyclooxygenase active site of COX-2 form salt bridges with Arg-120 and hydrogen bond with the phenolic oxygen of Tyr-355 (8). A major difference between COX-1 and COX-2 is how it is expressed and their consequent biological roles. COX-1 is typically constitutively expressed while expression of COX-2 tends to be induced. Therefore, COX-1 is seen to have more of a homeostatic function while COX-2 is more associated with inflammation and disease. Also, COX-2 has a wider specificity than COX-1, so it can bind substrates that cannot bind to COX-1. This is because COX-2 has a larger cyclooxygenase active site due to the differences in intermolecular distance of Tyr-355, Arg-120, Tyr-385, Ser-530, Ile-523 (Val in COX-2), Phe-518, Leu-352, Trp-387, Tyr-348, Gln-192, His-90, and His-513 (Arg in COX-2) (9). Although very similar, COX-1 and COX-2 cannot be replaced for one another – both are essential for biological functioning.
Overall, COX-1/Prostaglandin H2 Synthase is an incredibly important dimerized protein for maintaining tissue homeostasis. Its functioning is integral to the production of prostaglandins which are necessary for not only homeostasis but also inflammation and disease progression. By figuring out ways to more specifically target COX-1, potential treatments for diseases like cardiovascular disease could be found.