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

Domenico Paludi,Beatrice Dragani,Bengt Mannervik,Gun Stenberg,Antonio Aceto,Daniela Rossi Principe,Roberta Cocco

doi:10.1074/jbc.m104057200

Domenico Paludi, Beatrice Dragani + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m104057200

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Abstract

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction 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 interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Highlights

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST)
A previous sequence alignment study [18] revealed the presence of a second structural motif conserved at the N terminus of the ␣6-helix of all known GSTs. This motif corresponds to the specific (i, iϩ5) hydrophobic interaction, named the hydrophobic staple motif, formed between residue NЈ, located before the first helical residue (Ser154 in GSTA1-1, N-cap), and a residue N-4, located at position four within the helix
An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known GSTs

Summary

EXPERIMENTAL PROCEDURES

Materials—Wild-type human GSTA1-1 was obtained by expression of a cloned cDNA in Escherichia coli XL-1 Blue (Stratagene, La Jolla, CA) as previously described [29]. Temperature Dependence of Refolding in Vitro for Wild-type and Hydrophobic Staple Mutants—When the refolding of human GSTA1-1 and its mutants was to be monitored, 10 ␮M enzyme was first denatured in 4 M guanidinium chloride (GdnHCl) (0.2 M phosphate, 1 mM EDTA, 5 mM DTT, pH 7.0) at 25, 32, 40, and 45 °C for 30 min and diluted (defining time 0) 1:40 into renaturation buffer (0.2 M phosphate, 1 mM EDTA, 5 mM DTT, pH 6.5) at the same temperature. We have assumed: (i) that the enzyme reactivation follows, kinetically, a twostate model without intermediates so that the refolding rate, measured under native-like conditions, can be used to characterize the transition state for folding; (ii) that none of the mutations have any significant effect on the folding pathway, at least not when analyzed at subphysiological temperature. Figures were generated by using the RasMol (v2.6, 1994 –1996, Roger Sayle) program

RESULTS

NDc NDb mM

NDa NDa

DISCUSSION