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 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.
Rossjohn
J
J
The Ian Potter Foundation Protein Crystallography Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.
McKinstry
W J
WJ
Oakley
A J
AJ
Parker
M W
MW
Stenberg
G
G
Mannervik
B
B
Dragani
B
B
Cocco
R
R
Aceto
A
A
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
D005982:Glutathione Transferase