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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...

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ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (⌬pH) and the electrical potential differences (⌬⌿) across membranes. The optimum rate of synthesis reached 85 s ؊1 , and the H ؉ /ATP ratio of 4.0 ؎ 0.1 was obtained. ATP was synthesized at a considerable rate solely by ⌬pH, indicating ⌬⌿ was not absolutely required for synthesis. Consistent with the H ؉ /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. ATP synthase ͉ rotary motor ͉ membrane protein ͉ bioenergetics ͉ two-dimensional crystal M embers of the F o F 1 and V-ATPase superfamily (rotary ATPase/synthase) use a rotary catalytic mechanism to perform their specific function (1, 2). The F o F 1 mainly catalyzes ATP synthesis in mitochondria, chloroplasts, and aerobic bacteria (3, 4). In contrast, V-ATPases exist in the endomembranes of all eukaryotic cells and in the plasma membrane of some specific eukaryotic cells functioning as a proton pump with a variety of cellular functions (2). The homologues of eukaryotic V-ATPases are also found in the plasma membrane of some bacteria (5, 6). Like the F o F 1 , V-ATPase from the thermophilic eubacterium Thermus thermophilus catalyzes ATP synthesis (7,8). In addition, it has the simplest known subunit structure (Fig. 1a) and is thus an excellent model for studying the mechanism of action of these important molecules. Subunits A and B of V-ATPase are the counterparts of subunits ␤ and ␣ of F o F 1 ATPase. Three copies of each subunit are arranged around the central rotor, which is made of single copies of subunits D and F. The A 3 B 3 DF moiety, termed V 1 , is responsible for the ATP hydrolysis or ATP synthesis reaction. The remaining subunits, V o -a (sometimes referred to as subunit I), V o -c (sometimes referred to as subunit L), and V o -d, E, and G form the V o domain of T. thermophilus V-ATPase (9). The V o -c subunits, which are folded into two transmembrane helices, constitute a membraneembedded oligomeric ring structure (10). The V o -c rotor ring and subunit V o -a form a proton channel, as seen in the c rotor ring of the F o -a subunit, despite low sequence similarity between the proteins.The basic mechanism of ATP synthesis for F o F 1 is well understood, as described below. Briefly, the ring of the F o -c subunit oligomer and ␥-subunits of F 1 comprise the central rotor, and together these rotate as a single body (11). The transmembrane electrochemical potential gradient of proton [⌬ H ϩ ϭ PMF ϫ F (PMF, proton motive force; F, Faraday constant)] drives rotation of the roto...