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

Abstract

Prostaglandin E 2 (PGE 2 ) is a key mediator in inflammatory response. The main source of inducible PGE 2 , microsomal PGE 2 synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (MembraneAssociated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design.membrane protein | X-ray crystallography | enzyme mechanism P rostaglandins are potent lipid messengers and are involved in numerous homeostatic biological functions [for a review of eicosanoid biology, see review by C. D. Funk (1)]. They are enzymatically derived from the essential fatty acid arachidonic acid and the synthesis proceeds via the formation of prostaglandin H 2 (PGH 2 ), a reaction catalyzed by the constitutively active cyclooxygenase COX-1 and the inducible cyclooxygenase COX-2. PGH 2 acts as a substrate for a range of terminal prostaglandin synthases, including the PGE synthases (PGES, EC 5.3.99.3) that convert PGH 2 to PGE 2 .Microsomal prostaglandin E 2 synthase-1 (mPGES-1), colocalized and up-regulated in concert with COX-2, is the major source of inducible PGE 2 and is associated with inflammation and pain (2). Several studies support a role for mPGES-1 also in cancer cell proliferation and tumor growth (3). Because treatment with COX-2 selective inhibitors is associated with elevated cardiovascular risk, safer approaches involving, for example, PGE 2 reduction, are needed (4). Mice deficient in mPGES-1 have shown significantly reduced effect on hypertension, thrombosis, and myocardial damage compared with inhibition or disruption of COX-2, suggesting mPGES-1 to be a potential target for pharmaceutical intervention in various areas of diseases (2, 5).mPGES-1 belongs to a superfamily of Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism, the MAPEG family (6). Members of the MAPEG family can be found in prokaryotes and eukaryotes but not in archaea (7). The most closely related MAPEG member is the microsomal glutathione transferase-1...

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

“…This method generated significant improvements in the quality indicators for PDB ID codes 4AL0 and 4AL1 [1.16 and 1.95 Å, respectively (13)] and revealed a ligand-dependent contact network that corroborates the mechanism suggested from biochemical data. These van der Waals interactions within the binary complex with GSH (PDB ID code 4AL0) reveal an extensive network of correlated side-chain motions within the cytoplasmic "C-domain" that forms the bottom of the active site and confirm a dynamic role of Asp-49 in catalysis.…”

“…Subsequent analysis of the structure with CONTACT revealed an extensive network of correlated side chain interactions centered upon the short, cytoplasmic helix separating transmembrane helices I and II that was referred to by Sjögren et al (13), and will be hereafter, as the C-domain. Of most interest is that the network facilitates signal transduction from residues involved in the recognition of GSH to the active site residue Asp-49.…”

“…S4. We submitted the PDB entries associated with the recently published crystal structure of mPGES-1 (PDB ID codes 4AL0 and 4AL1) (13) to the qFit server (smb.slac.stanford.edu/qFitServer/) (20). This software automatically samples conformational heterogeneity that is interpretable by fitting partial occupancy conformational ensembles into low-level electron density.…”

“…We have previously proposed that this conserved arginine residue is also essential for enzymatic activity in mPGES-1 as Arg-126 (12). The recent structural determination of mPGES-1, however, at an exceptionally high resolution of 1.16 Å, uncovered several unanticipated structural features (13). The active sites, found at the three monomeric interfaces, show that Ser-127 is positioned near the GSH thiol group, indicating that it may act as a hydrogenbond donor to assist in thiolate formation during catalysis.…”

“…We found that Ser-127 is nonessential for catalysis, whereas Arg-126 and Asp-49 are crucial and mutually dependent for native isomerase activity of the enzyme. Because the latter codependence of activity could be rationalized by a dynamic functional role of these residues, we turned to the highresolution X-ray data (13,15) deposited in the Protein Data Bank (PDB; www.rcsb.org) (16) to provide evidence of their dynamic motion within the crystal structure. Several recent studies have shown that the information present in such data is often underestimated and that it is possible to refine multiple conformations of residues simultaneously, each with individually refined occupancy and B factors, without overfitting the data (17)(18)(19)(20)(21)(22).…”