Structures of thermolabile mutants of human glutathione transferase P1-1

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the b eginning of helix alpha 6 in the core of virtually all glutathione transfer ases (GST) and GST-related proteins. It has been demonstrated that this loc al motif is important in determining the alpha-helical propensity of the is olated alpha 6-peptide and plays a crucial role in the folding and stabilit y of GSTs. Its removal by site-directed mutagenesis generated temperature-s ensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A ), in which the capping residues have been substituted by alanine, have bee n generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permiss ive temperature, have been crystallized and their three-dimensional structu res determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stabi lity of the enzyme at higher temperatures upon single substitution of a cap ping residue. (C) 2000 Academic Press.