title: Structures of thermolabile mutants of human glutathione transferase P1-1.
abstract: An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive 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 been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures 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 stability of the enzyme at higher temperatures upon single substitution of a capping residue.
authors.affiliation: The Ian Potter Foundation Protein Crystallography Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Rossjohn
authors.forename: W J
authors.initials: WJ
authors.lastname: McKinstry
authors.forename: A J
authors.initials: AJ
authors.lastname: Oakley
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: G
authors.initials: G
authors.lastname: Stenberg
authors.forename: B
authors.initials: B
authors.lastname: Mannervik
authors.forename: B
authors.initials: B
authors.lastname: Dragani
authors.forename: R
authors.initials: R
authors.lastname: Cocco
authors.forename: A
authors.initials: A
authors.lastname: Aceto
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary
chemical_list: D005982:Glutathione Transferase