The N-subdomain of the thioredoxin fold of glutathione transferase is stabilised by topologically conserved leucine residue
Date
2013-04-30
Authors
Khoza, Thandeka Ntokozo
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Abstract
The thioredoxin-like (Trx-like) fold is preserved in various protein families with diverse
functions despite their low sequence identity. Glutathione transferases (GSTs) are
characterised by a conserved N-terminal domain with a thioredoxin–like βαβαββα secondary structure topology and an all alpha-helical domain. GSTs are the principal phase II enzymes involved in protecting cellular macromolecules from a wide variety of reactive electrophilic compounds. It catalyses the conjugation of reduced glutathione (GSH) to an electrophilic substrate to form a hydrophilic and non-toxic compound. The binding site for GSH (G-site) is located in the N-terminal domain of GSTs. The sequence identity within members of the
Trx-like superfamily is low; however, the members of this family fold into a conserved
βαβαββα topology. It, therefore, seems reasonable that there are topologically conserved
residues within this fold whose main role is to drive folding and/or maintain the structural
integrity of the Trx-like fold. Structural alignments of the N-subdomain (βαβ motif) of the
GST family shows that Leu7 in β1 and Leu23 in α1 are topologically conserved residues.
The Leu7 side chain is involved in the packing of α1β1α2 and α3, whilst Leu23 is mainly
involved in van der Waals interactions with residues in α1 and the loop region connecting α1
and β2. Taking into account the types of interaction that both Leu7 and Leu23 are involved
in, as well their location in close proximity to the G-site, it was postulated that both these
residues may play a role in the structure, function and stability of the GST family of proteins.
Leu7 and Leu23 are not directly involved in the binding of GSH but they could be important
in maintaining the G-site in a functional conformation via correct packing of the Nsubdomain.
The homodimeric human class Alpha of GST (hGSTA1-1) was used as the representative of
the GST family to test this hypothesis. The bulky side chains of Leu7 and Leu23 were
replaced with a less bulky alanine residue to prevent altering the hydrophobicity of the βαβ
motif. The effect of the mutation on the structure, function and stability of hGSTA1-1 was,
therefore, studied in comparison with the wild-type using spectroscopic tools, X-ray
crystallography, functional assays and conformational stability studies.
The impact of the mutations on the structure of the enzyme was determined using
spectroscopic tools and X-ray crystallography. The X-ray structures of the L7A and L23A
mutants were resolved at 1.79 Å and 2.2 Å, respectively. Analysis of both X-ray structures
shows that the mutation did not significantly perturb the global structure of the protein, which
correlates with far-UV CD and intrinsic fluorescence spectroscopic data. In addition,
structural alignments using the C-alpha gave root mean square deviation (r.m.s.d) values of
0.63 Å (L7A) and 0.67 Å (L23A) between the wild-type and mutant structures. However,
both the L7A and L23A structures showed the presence of a cavity within the local
environment of each mutation. The functional properties of the mutants were also similar to
those of the wild-type as determined by specific activity and 8-anilino-1-naphthalene sulfonate
(ANS)-binding, indicating that Leu7 and Leu23 are not involved in the function of hGSTA1-
1.
The conformational stability of L7A and L23A proteins was probed using thermal-induced
unfolding, pulse proteolysis and urea-induced equilibrium unfolding studies. The thermal
stability of L7A and L23A hGSTA1-1 was reduced in comparison to the wild-type protein.
This was consistent with proteolytic susceptibility of L7A and L23A proteins which indicates
that both mutants are more prone to thermolysin digestion when compared to wild-type
hGSTA1-1. This also correlates with urea-induced equilibrium studies. The ΔG(H2O) value
(23.88 kcal.mol-1) for the wild-type protein was reduced to 12.6 and 10.49 kcal.mol-1 in L7A
and L23A hGSTA1-l, respectively. Furthermore, the m-values obtained for the L7A and
L23A proteins were 1.46 and 1.06 kcal.mol-1.M-1 urea, respectively; these were much lower
than that obtained for the wild-type protein (4.06 kcal.mol-1.M-1 urea). The low m-values
obtained for the mutant proteins indicated that the cooperativity of hGSTA1-1 unfolding was
significantly diminished in both mutations. The results obtained in this study indicate that the
topologically conserved Leu7 and Leu23 in the N-subdomain of hGSTA1-1 play a crucial
role in maintaining the structural stability of the thioredoxin-like domain and are not involved
in the function of the enzyme.
Description
A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg,
in fulfillment of the requirements for the degree of Doctor of Philosophy.
Johannesburg, 2012