The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif

An N-capping box and a hydrophobic staple motif are strictly conserved in t he core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been gener ated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicat e that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabili zing than the first helical residue S154A mutation (N-cap) suggests that th e appropriate conformation of the conserved substructure formed by the alph a6-helix and preceding loop (GST motif II) is crucial for the overall prote in stability. The refolding study of GSTAI-1 variants supports the predicti on that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interact ion favors essential inter-domain contacts and, thereby, in contrast to cap ping interactions, accelerates the enzyme reactivation. Its strict conserva tion in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.