Structural basis for induced formation of the inflammatory mediator prostaglandin E2.

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 PGE(2). 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.

Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1.

Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

Structural basis for detoxification and oxidative stress protection in membranes.

Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 A resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

Two-dimensional crystallization and electron crystallography of MAPEG proteins.

Members of the membrane-associated proteins in the eicosanoid and glutathione metabolism (MAPEG) superfamily have been subjected to two-dimensional crystallization experiments. A common denominator for successful attempts has been the use of a low lipid/protein ratio in the range of 1-9 (mol/mol). Electron crystallography demonstrated either hexagonal or orthorhombic packing of trimeric protein units. Three-dimensional structure analysis of the MAPEG member microsomal glutathione transferase 1 has shown that the monomer for this protein contains a left-handed bundle of four transmembrane helices. It is likely that this is a common structural motif for MAPEG proteins, because projection maps of all structurally characterized members are very similar.

Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 1.

Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.