Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study.
The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic alpha-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1.S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand-protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex.
Kuhnert
Diane C
DC
Protein Structure-Function Research Programme, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Sayed
Yasien
Y
Mosebi
Salerwe
S
Sayed
Muhammed
M
Sewell
Trevor
T
Dirr
Heini W
HW
D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D011522:Protons; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry; D014867:Water / Q000737:chemistry
D007527:Isoenzymes; D011522:Protons; D012997:Solvents; D007532:Isoleucine; D014867:Water; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005978:Glutathione; C033550:hexylglutathione