Class Pi Glutathione Transferase Unfolds via a Dimeric and Not Monomeric Intermediate: Functional Implications for an Unstable Monomer

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (INK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Common. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.