The conserved N-capping box in the hydrophobic core of glutathione S-transferase P1-1 is essential for refolding. Identification of a buried and conserved hydrogen bond important for protein stability.
The second domain of cytosolic glutathione S-transferases (GSTs) contains a strictly conserved N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) at the beginning of alpha6-helix in the hydrophobic core of the molecule. Considering the specific function attributed to capping box residues in the helix nucleation, we decided to investigate, by site-directed mutagenesis, the role that this motif could have in the folding and stability of human GSTP1-1. Both capping box mutants, S150A and D153A, were significantly more thermolabile than wild-type GSTP1-1, indicating that the local destabilization of the alpha6-helix determined by a single capping residue mutation affects the overall stability of the protein. The results also show that, in addition to capping interactions, an important role in the stability of the final structure of the protein is played by a buried and conserved hydrogen bond formed between the side chain of Asp-153 and the amide NH of Ile-144 located in the long loop preceding alpha6-helix. Reactivation experiments in vitro indicate that the N-capping box is essential for refolding of the denatured protein at a physiological temperature. The results suggest that during folding this buried and conserved motif, making a definite set of native-like contacts, determines the formation of a specific folding nucleus that probably represents a transition state of the folding process.
Dragani
B
B
Dipartimento di Scienze Biomediche, Università "G. D'Annunzio," Via dei Vestini, 31, 66100 Chieti, Italy.
Stenberg
G
G
Melino
S
S
Petruzzelli
R
R
Mannervik
B
B
Aceto
A
A
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D013696:Temperature
D007527:Isoenzymes; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase