Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of Ϸ50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4 -ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal structures of human CYP3A4 in complex with two well characterized drugs: ketoconazole and erythromycin. In contrast to previous reports, the protein undergoes dramatic conformational changes upon ligand binding with an increase in the active site volume by >80%. The structures represent two distinct open conformations of CYP3A4 because ketoconazole and erythromycin induce different types of coordinate shifts. The binding of two molecules of ketoconazole to the CYP3A4 active site and the clear indication of multiple binding modes for erythromycin has implications for the interpretation of the atypical kinetic data often displayed by CYP3A4. The extreme flexibility revealed by the present structures also challenges any attempt to apply computational design tools without the support of relevant experimental data.drug metabolism ͉ structural flexibility ͉ x-ray crystallography ͉ inhibitor ͉ substrate C ytochrome P450 3A4 (CYP3A4) is the most abundant of the xenobiotic-metabolizing CYP isoforms, and interactions with CYP3A4 must always be taken into consideration during the development of new medications (1).In recent years, a number of structures of mammalian CYP isoforms, including CYP2C5 (2), CYP2C9 (3, 4), CYP2C8 (5), CYP2B4 (6), CYP2A6 (7), and CYP3A4 (8, 9) have been solved. All of these structures were determined from modified versions of the protein where the N-terminal transmembrane helix was truncated and, in some cases, a number of mutations aimed at increasing solubility were introduced. The mammalian CYP structures all adopt the general CYP fold first described in 1987 when the structure of the bacterial P450 CYP101 was determined by x-ray crystallography (10).The ligand-free structure of CYP3A4 was published in 2004 by two independent groups (8, 9). These structures are very similar (11). The most remarkable features are the short F and G helices (nomenclature adapted from Poulos et al.; ref. 10) and a large, highly ordered hydrophobic core of phenyl alanine residues above the active site (8, 9). CYP3A4 is known to metabolize large substrates such as bromocriptine (M r 655 Da) and cyclosporine (M r 1,203 Da). A number of studies also indicate that CYP3A4 displays ligand binding that does not follow Michaelis-Menten type kinetics, and it has been suggested that two or more ligand molecules can bind to the CYP3A4 active site simultaneously (12-15). In light of these observations, the volume of the active site in the published ligand-free structures is smaller than expected, comparable with the active-site cavity seen in CYP2C9 and CYP2C8, which led Williams et al. (8) to speculate that conformational changes may o...

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...

The characteristics of CD8+ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8+ T cell populations. In most experimental systems, heterogeneous populations of primed CD8+ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8+ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8+ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8+ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8+ T cells were compared in vitro. We found that CD8+ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8+ T cells differ from naive CD8+ T cells. These primed hyperresponsive CD8+ T cells might play an important role in the memory response.

Background:The enzyme myeloperoxidase produces chlorine bleach at sites of inflammation. Results: 2-Thioxanthines are potent mechanism-based inactivators of myeloperoxidase. Conclusion: 2-Thioxanthines block production of chlorine bleach during inflammation. Significance: Mechanism-based inactivators of myeloperoxidase should limit oxidative stress at sites of inflammation.

Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 Å . The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptidebased drug design.