Journal of Molecular Biology
Structural Basis for Detoxification and Oxidative Stress Protection in Membranes
Introduction
Oxidative stress and exposure to toxic compounds are constant threats to living organisms. Efficient protection systems involving specific enzymes have emerged throughout evolution. Glutathione transferases (GSTs) are playing a crucial role in cellular biotransformation of electrophilic compounds through binding and positioning the tri-peptide glutathione (GSH), γ-l-glutamyl-l-cysteinyl-glycine, for nucleophilic attack. Based on sequence and structural similarity distinct, ancient glutathione transferase protein families of cytoplasmic, microsomal, mitochondrial and bacterial origin have been identified.1 In addition to the detoxification role played by GSTs, homologous members within the families may carry out distinctly different functions. Soluble GSTs from mammals, plants, bacteria and insects have been well characterised structurally and subdivided into several classes. The canonical cytosolic enzymes are dimers and related by evolution to glutaredoxin having the unique thioredoxin βαβαββα fold.2 Here we describe the first detailed structure of a GST from the microsomal family, MGST1 (microsomal glutathione transferase 1). Like most soluble GSTs, MGST1 catalyses conjugation of GSH to a number of electrophilic compounds and is therefore playing an important role in biotransformation of xenobiotic compounds.3 In addition, MGST1 protects biological membranes from degradation through GSH-dependent reduction of unspecifically peroxidised phospholipids.4 The microsomal GSTs, more recently termed MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism), also include members that are crucial for synthesis of mediators of fever, pain and inflammation.5,6 These pathophysiological responses are regulated by GSH-dependent transformations of specific oxidised lipid intermediates, prostaglandin H2 endoperoxide and leukotriene A4 epoxide, to prostaglandin E2 and leukotriene C4, respectively. Thus, variations of a hitherto unknown common protein structure have emerged to selectively catalyse distinct activities with strong physiological and pathophysiological significance. In the present work the structural basis for these reactions has been elucidated. By determining the atomic structure of a key enzyme we now begin to understand the specificity and function of these proteins. As they are potential targets for treatment of common diseases such as rheumatoid arthritis and asthma, the results should impact on drug development.
Section snippets
Structure of MGST1
Upon reconstitution into lipid bilayers at low lipid/protein ratio MGST1 forms two-dimensional crystals of two different two-sided plane groups, p22121 and p6, both suitable for analysis by electron crystallography. We have now solved the structure of the rat enzyme to 3.2 Å in plane resolution (4.0 Å perpendicular to the membrane plane) using data from both crystal forms (Figure 1(a) and Table 1). As previously described7,8 both the p6 and the p22121 maps contain densities with three repeats
Discussion
A fundamental aspect of glutathione transferase catalysis involves the stabilisation of the reactive nucleophilic thiolate anion form of GSH. The presence of the GSH thiolate has been demonstrated by spectroscopy in both soluble glutathione transferases26 and MGST1.27 In soluble glutathione transferases a tyrosine or serine hydroxyl has been shown to hydrogen bond to the GSH thiolate and thereby lower its pKa value by several units to ≈6.28 In MGST1 GSH is surrounded by several residues that
Specimen preparation
MGST1 was purified from rat liver microsomes3 and two-dimensional crystals with p6 and p22121 two-dimensional plane group symmetry were prepared by reconstitution into phospholipid bilayers by dialysis at a molar lipid to protein ratio varying between 3 and 5 as previously.36 The quality of the reconstituted crystals was evaluated by electron microscopy of negatively stained specimens. Excellent crystal preparations were selected for cryo-electron microscopy. These specimens were prepared on
Acknowledgements
We thank Gerd Lundqvist and Gudrun Tibbelin for excellent technical assistance. This work was supported by grants from Swedish Research Council (to H.H.) and from the Swedish Cancer Society, the Swedish National Board for Laboratory Animals and Funds from Karolinska Institutet (to R.M.). P. J. H. has been a recipient of a fellowship from the Swedish Foundation for Strategic Research.
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Present address: P.J. Holm, Aarhus University, Department of Molecular Biology, Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark.
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Present address: K. Mitsuoka and N. Gyobu, Structural Analysis Team, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi 2-41-6, Koto-Ku, Tokyo, 135-0064, Japan.