Structural basis for induced formation of the inflammatory mediator prostaglandin E ₂

Abstract

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH 2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH 2 to PGE 2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein M icrosomal prostaglandin E synthase 1 (MPGES1) is the key enzyme in pathology related production of PGE 2 from cyclooxygenase (Cox) derived PGH 2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxygenase activating protein (FLAP), leukotriene C 4 synthase (LTC4S), microsomal glutathione transferase (MGST)1, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES known and catalyzes the oxidoreduction of prostaglandin endoperoxide H 2 into PGE 2 with an apparent k cat /K m of 310 mM Ϫ1 s Ϫ1 [supporting information (SI) Fig. S1]. The enzyme equally well catalyses the oxidoreduction of endocannabinoids into prostaglandin glycerol esters (4) and PGG 2 into 15-hydroperoxy-PGE 2 (5). In addition, the enzyme confers low glutathione transferase and glutathione-dependent peroxidase activities (5). The biological significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1.MPGES1 protein expression levels are in most cases low, and proinflammatory stimuli induce its cellular expression and activity, which is prevented by corticosteroids (1, 6-8). The predominant source of PGH 2 seems derived from Cox-2, although Cox-1 may also contribute (9). Studies, mainly from disruption of the MPGES1 gene in mice, indicate key roles for MPGES1-generated PGE 2 in pathological conditions such as chronic inflammation, pain, fever, anorexia, atherosclerosis, stroke and tumorigenesis (10). Recently, a role for MPGES1 in regulating neonatal respiration was described in ref. 11. MPGES1 has been shown to be overexpressed in rheu...

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

“…A putative structure of an open conformation of the active site, with a critical Arg residue (Arg-126), was obtained through modeling against the crystal structure of human LTC4S. Hence, the mPGES-1 structure presented by Jegerschöld et al (20) agrees very well with the results of our own investigation and suggests that further mechanistic studies must take into account the consequences of protein dynamics.…”

“…During the course of these investigations, Jegerschöld et al (20) reported a structure of mPGES-1 at 3.5 Å resolution, obtained by electron crystallography. GSH was found to bind in a U-shaped conformation, and, interestingly, the authors could not find an access path for PGH 2 to the bound GSH molecule, indicating that the protein was in a closed conformation and that dynamic changes, involving helices 1 and 4, occur at the active site during binding and turnover of the lipid substrate.…”

Crystal structure of microsomal prostaglandin E2synthase provides insight into diversity in the MAPEG superfamily

“…The structure differs significantly from the previously reported electron crystallography structure of mPGES-1 (14). In particular, glutathione binding and coordination are different in the two structures.…”

“…This was later confirmed by the X-ray crystal structures of FLAP (10) and LTC 4 synthase (11,12). There are also low-resolution 3D electron crystallography structures of MGST1 and mPGES-1 (13,14). However, these structures did not allow for detailed structural analysis, and concerns regarding the accuracy of the MGST1 structure have been raised (15).…”

Calcium/calmodulin‐dependent protein kinase II regulates cyclooxygenase‐2 expression and prostaglandin E2 production by activating cAMP‐response element‐binding protein in rat peritoneal macrophages

“…3,4 Among these factors, prostaglandin E 2 (PGE 2 ) is an essential mediator that contributes to vasodilatation, pain and fever. 5,6 Additionally, PGE 2 is also overproduced in many tumours, where it aids cancer progression by promoting angiogenesis and metastasis. 7,8 In macrophages, PGE 2 production is tightly controlled by arachidonic acid (AA) release and cyclooxygenase-2 (COX-2) expression.…”