The stability of monomeric class Mu GST M1

Abstract

Cytosolic glutathione transferases (GSTs) are obligate homo- or heterodimers with a complex canonical structure whose assembly involves interactions at the domain interface and at the subunit interface. Each subunit is composed of an N-terminal GSH binding domain (domain 1) and a C-terminal all-α-helical domain (domain 2). Currently, little is known about the stability, folding and assembly of GST subunits in the absence of quaternary interactions. The stabilities of several GST dimers, including class Mu GST, are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits, namely, a hydrophobic lock-and-key motif and a buried charge cluster motif. In class Mu GST M1-1 from rat, the mutation of the two key residues , Phe-56 and Arg-81, involved in subunit-subunit interaction motifs, into Ser and Ala, respectively, has been reported to result in a stable monomer (Thompson et al. (2006) Biochemistry 45, 2267- 2273). In this study, using gel filtration chromatography, it was confirmed that simultaneous disruption of both motifs in rGST M1-1 to create the F56S/R81A mutant results in complete dissociation of the dimer to a monomeric protein. Comparative studies reveal the monomeric mutant to be catalytically inactive due to the loss of the subunitsubunit contacts, displaying approximately 2.7 % activity relative to the wild type enzyme. Moreover, the stability and folding of a monomeric form of rGST M1-1 were investigated under equilibrium conditions by circular dichroism, intrinsic tryptophan fluorescence, and ANS binding. The results show that the monomer is less stable, with a ΔG(H2O) value of 13.3 kcal/mol relative to the reported ΔG(H2O) value of 16.5 kcal/mol (Hornby et al. (2000) Biochemistry 39, 12336-12344) for the second transition (i.e. I U) of the dimeric wild type enzyme.