Influence of the dimer interface on glutathione transferase structure and dynamics revealed by amide H/D exchange mass spectrometry

Mammalian glutathione (GSH) transferases are dimeric proteins, many of which share a common hydrophobic interaction motif that is important for dimer stability. In the rGSTM1-1 enzyme this motif involves the side chain of F56, located on the 56 loop of the N-terminal domain, which is intercalated between the alpha 4- and alpha 5-helices of the C-terminal domain of the opposing subnuit. Disruption of the complementary interactions in this motif by mutation of F56 to serine, arginine, or glutamate is known to have deleterious effects on catalytic efficiency but remarkably different effects on the stability of the dimer [Hornby et al. (2002) Biochemistry 41, 14238-14247]. The structural basis for the behavior of the mutants by amide H/D exchange mass spectrometry is described. A substantial decrease in H/D exchange is observed in the GSH binding domain and in parts of the dimer interface upon substrate binding. The F56S and F56R mutants exhibit enhanced H/D exchange kinetics in the GSH binding domain and at the dimer interface. In contrast, the F56E mutant shows a decrease in the rate and extent of amide H/D exchange at the dimer interface and enhanced exchange kinetics in the GSH binding domain. The results suggest that the F56E mutant has a restructured dimer interface with decreased solvent accessibility and dynamics. Although all of the F56 mutations disrupt the GSH binding site, the effects of the mutations on the structure of the subunit interface and dimer stability are quite distinct.