Structural and functional studies of microsomal glutathione transferase 1
Sammanfattning: The microsomal glutathione transferase 1 (MGST1) is present in high amounts in the endoplasmic reticulum (ER) as an integral homo-trimeric membrane protein with a molecular weight of 51.9 kDa. MGST1 possess glutathione transferase and peroxidase activity and protects accordingly against xenobiotic toxic substances and lipid peroxidation making it interesting in cancer, heart and vascular disease prevention and ageing related disorders. MGST1 belong to the super family of Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism ~PEG) together with the two other microsomal glutathione transferases 2 and 3, microsomal prostaglandin E synthase 1 (MPGES1), leukotriene C4 synthase and 5-lipoxygenase activating protein. This thesis describes results from structural and functional studies of MGST1 purified from rat fiver microsomes. For the structural studies the purified protein was reconstituted into lipid bilayers forming large well-ordered two-dimensional crystals of two different symmetries (p22121 and p6) suitable for electron crystallography. We have determined the structure to 3.5Å resolution from both crystal forms allowing building of an atomic model thus making MGST1 the first enzyme out of six membrane proteins ever solved to near-atomic resolution by electron crystallography. MGST1 consists of three identical subunits, each consisting of a left handed four helix bundle comprising the transmembrane domain. In addition a cytosolic domain probably made up of four beta strands links the transmembrane helices 1 and 2. Helices 2 and 3 are connected in the ER lumen by a tripeptide loop conserved between MGST1 and the closest MAPEG relative MPGES1. Helices 3 and 4 are connected on the cytosolic side of the membrane by a rigid proline rich loop. Previously noted similarity to subunit 1 of cytochrome c oxidase was confirmed by structural alignment with MGST1 thus indicating a common evolutionary origin. MGST1 binds a hydrophobic second substrate to which reduced glurathione (GSH) is conjugated. To characterise the hydrophobic binding site regarding stoichiometry and affinity of hydrophobic substrate molecules to MGST1, inhibitory assays and equilibrium dialysis were performed. The most interesting results were obtained with the standard assay product GS13N13 which demonstrated that product inhibition of MGST1 occurs and that the binding stoichiometry is three per trimer. The latter is in agreement with unpublished observations from ESI-mass spectrometric analysis showing the occurrence of three GSH molecules bound to the MGST1 trimer. Investigations of cavities in the structure revealed a possible substrate binding pocket between helices 1 and 2 of one subunit and helix 2 of a neighbouring subunit. Atomic force microscopy measurements revealed the vertical dimension of the 2D crystals. Analysis of time-resolved exchange of the amide backbone proton for deuterium detected by mass spectrometry (H/D exchange) was performed on GSH bound and apo-MGST1 both in activated and unactivated form in detergent and proteoliposomes respectively. These studies showed that the cytosolic domain of MGST1 harbours the glutathione binding site. In addition it is suggested that the binding of GSH to MGST1 induces a cooperative conformational transition possibly involving movements of the transmembrane helices and a reordering of the cytosolic domain consistent with observations from the structural investigation. Alkylation of the stress sensor cysteine-49 preorganises the structure and facilitates the cooperative transition resulting in catalytic activation. Importantly, the enzyme reconstituted into phospholiposomes behaved in a similar fashion as the detergent solubilised enzyme regarding exchange dynamics indicating the validity of using detergent complexes in functional studies of the native enzyme.
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