Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione.

The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques.

Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release.

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies.

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.

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.

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.

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.

The 3-D structure of microsomal glutathione transferase 1 at 6 A resolution as determined by electron crystallography of p22(1)2(1) crystals.

Pure solubilised microsomal glutathione transferase 1 (MGST1) forms well-ordered two-dimensional (2-D) crystals of two different symmetries, one orthorhombic (p22(1)2(1)) and one hexagonal (p6), both diffracting electrons to a resolution beyond 3 A. A three-dimensional (3-D) map has previously been calculated to 6 A resolution from the hexagonal crystal form. From orthorhombic crystals we have now calculated a 6 A 3-D reconstruction displaying three repeats of four rod-like densities.