Structural analysis of microsomal glutathione transferase 1 by electron crystallography

Abstract: The goals of this project were the two-dimensional crystallization of the membrane protein microsomal glutathione transferase 1 and the three-dimensional structure determination by electron crystallography. Microsomal glutathione transferase 1 is a homotrimeric membrane protein. The enzyme plays an important role in the detoxication in liver, where microsomal glutathione transferase 1 conjugates glutathione to a multitude of hydrophobic electrophiles. It was found that microsomal glutathione transferase 1 can be induced to form twodimensional crystals. These crystals are grown by reconstitution into phospholipid bilayers by detergent removal through dialysis. The protein forms p21212 crystals after six days of dialysis when it is reconstituted in the presence of three phospholipid molecules per protein trimer. Fractional changes below this lipid-to-protein ratio result in a p6 crystal type. The longevity of the crystals can be improved by increasing the dialysis time to eight days. Grown at room temperature (21º C and with an unusually high initial detergent concentration of 1% Triton X-100, the two-dimensional crystals extend to at least 3-10 [my]m. Furthermore, conclusions could be drawn about the rate-dependence of the crystal formation. Both of these crystal forms are ordered to beyond 3 Å resolution as judged by electron diffraction of untilted specimens. Initially a projection map of the p21212 crystals was calculated from images at a resolution of 4 Å. [alpha]-helical secondary structure was apparent in the center of the trimer, but an outer protein density belonging to each monomer could not be identified. An additional projection map of the p21212 crystal form was calculated of images as well as electron diffraction patterns at a resolution of 3 Å, which revealed an additional a-helical density in the outer protein domains of the trimer as well as an unidentified density. The protein packing in the projection map of the p6 unit cell, calculated at 3 Å, showed identical elements of protein packing in both crystal forms. By comparing the projection structures of both crystal types, densities indicating ordered lipid headgroups or amino acid side chains could be identified. The three-dimensional structure of the p6 crystal form was calculated to a resolution of 6 from electron diffraction patterns and images with tilt angles to 60º. The three-dimensional map shows a right-handed [alpha]-helical bundle. Three of the helices are nearly perpendicular to the plane of the lipid bilayer and parallel to each other. The fourth helix is highly tilted, and thus it could not be identified in the projection map. The four helices could be assigned. The N- and C-terminal extend into the lumen of the endoplasmic reticulum on the periphery of the trimer. Helix 2 and helix 3 are connected by a short loop, which also faces the lumenal side of the membrane. The helices 2-4 appear to be important in the monomer-monomer contacts within the trimer. Microsomal glutathione transferase 1 is a member of the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily. It was suggested that the [alpha]-helical bundle observed for microsomal glutathione transferase 1 is representative of the tertiary structures of other members of the MAPEG superfamily.