Biochemical and thermodynamic characterisation of ligand-binding to class alpha glutathione transferase A1-1
Date
2010-07-02T11:51:44Z
Authors
Sayed, Yasien
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Phenylalanine 51 (F51) in the human class alpha GST forms part of a hydrophobic lockand-
key intersubunit motif at the dimer interface. Protein engineering techniques were
used to replace the phenylalanine key with serine. The results indicated that the mutant
protein is dimeric with a native-like core structure indicating that F51 at the dimer
interface is not essential for dimerisation to occur. Replacing F51 with serine impacts on
the catalytic and ligandin function suggesting that tertiary structural changes have
occurred at/near the active and non-substrate ligand binding sites. The F51S mutant also
displays an enhanced exposure of hydrophobic surface as well as ligandin function. The
F51S mutant displays a diminished conformational stability when compared to the wildtype
protein. The lock-and-key intersubunit motif, therefore, although not essential for
dimerisation to occur does stabilise the quaternary structure at the dimer interface.
A unique structural feature of the class alpha GSTs is the C-terminal helix (residues 207-
221). In this study, the role of F221 was assessed by deleting it from the C-terminal helix
9 of hGSTA1-1. The results showed that the deletion of F221 does not affect the
secondary, tertiary and quaternary structure of the protein as observed using far-UV CD
measurements, enzyme activity and conformational stability as probes, respectively. The
wild-type protein binds ~ 1.7-fold more ANS than the F221del protein. Binding affinity
studies indicated that although both proteins bind ANS with the same affinity, the wildtype
protein binds ANS with a higher capacity than the F221del protein. ANS binding to
the wild-type and F221del proteins in the presence of urea (0 - 5.5 M urea) indicated that
F221 is required for stabilising helix 9 at the C-terminal of hGSTA1-1. Therefore, F221
is not required for catalysis nor does it impact on the conformational stability of the
protein. F221 does, however, affect the ligandin function and is required for the stability
of helix 9 at the C-terminus of hGSTA1-1.
ITC was used to dissect the binding energetics of glutathione (GSH) and glutathione
sulfonate (GSO3
-) to the wild-type and Y8F hGSTA1-1 proteins. The contribution of the
tyrosyl hydroxyl group to the binding of GSH and GSO3
- indicated that the Y8F mutant
binds GSH tighter than the wild-type protein and the wild-type protein, in turn, binds
GSO3
- tighter than the Y8F mutant protein. The Y8F mutant displays a larger negative
vi
DCp than the wild-type protein when complexed with either GSH or GSO3
-. This
indicates the burial of a larger solvent-exposed hydrophobic surface area for the Y8F
mutant than the wild-type protein. The burial of a large solvent-exposed hydrophobic
surface area is related to the immobilisation of helix 9 onto domain I in the presence of
active site ligands. The observation that the Y8F mutant displays burial of larger solventexposed
hydrophobic surface area suggests that the tyrosyl hydroxyl group controls the
dynamics of helix 9 at the C-terminal of hGSTA1-1. The DDG values also suggest that
the tyrosyl hydroxyl group stabilises the thiolate anion at the active site in the wild-type
protein.
The binding energetics of non-substrate ligands (ANS and BSP) to the wild-type human
class alpha GSTA1-1 were evaluated. The stoichiometry of the interaction between the
wild-type protein and ANS indicated that one molecule of ANS binds per protein
monomer. The binding interactions between ANS and the wild-type protein are
enthalpically favourable indicating the possibility of hydrogen bond formation. ANS
binding to the wild-type protein also resulted in the reduction of non-polar surface area
exposed to solvent. It is proposed that the ANS binding site is the region adjacent to
domain I that becomes buried when helix 9 is immobilised.
The binding of BSP to the wild-type protein involves a high and low affinity set of
binding sites. The high affinity binding site binds one molecule of BSP per protein
monomer whereas the low affinity site is capable of accommodating a minimum of ~ four
BSP molecules. The binding energetics to the high affinity site is both enthalpically and
entropically favourable with each term contributing favourably to the favourable Gibbs
free energy of binding. Binding to the lower affinity site is not very favourable
enthalpically and the major driving force behind the favourable Gibbs free energy of
association is the entropic factor. This interaction, therefore, appears to be entropically
driven.
Description
PhD, Faculty of Science (Molecular and Cell Biology), University of the Witwatersrand, 2001.
Keywords
biochemistry, thermodynamics