Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST...

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Bibliographic Details
Published in:	Scientific reports Vol. 7; no. 1; pp. 7897 - 10
Main Authors:	Kuang, Qie, Purhonen, Pasi, Ålander, Johan, Svensson, Richard, Hoogland, Veronika, Winerdal, Jens, Spahiu, Linda, Ottosson-Wadlund, Astrid, Jegerschöld, Caroline, Morgenstern, Ralf, Hebert, Hans
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 11-08-2017 Nature Publishing Group Nature Portfolio
Subjects:	101/28 45 45/70 631/45/607 631/535/1258/1259 82 82/80 Alanine Amino acids Animals Arginine Binding Sites Catalysis Conformation Crystallography Crystals Detoxification Electron diffraction Glutathione - chemistry Glutathione - metabolism Glutathione transferase Glutathione Transferase - chemistry Glutathione Transferase - genetics Glutathione Transferase - metabolism Humanities and Social Sciences Medicin och hälsovetenskap Microscopy, Electron, Transmission Models, Molecular multidisciplinary Mutagenesis Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - metabolism Phospholipids Protein Binding Protein Conformation Protein Multimerization Rats Science Science (multidisciplinary)
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Summary:	Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-017-07912-3