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Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

Wongsantichon J, Robinson RC, Ketterman AJ - Biosci. Rep. (2015)

Bottom Line: Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance.The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order.This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138673.

No MeSH data available.


Superposition of the histidine interface motif of Epsilon GSTs from four speciesThe residue numbering refers to the D. melanogaster GST Epsilon 6. The superimposed structures are 4YH2 (the present study, D. melanogaster), 2IMI (A. gambiae), 3ZML (A. funestus) and 3VWX (M. domestica).
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Figure 3: Superposition of the histidine interface motif of Epsilon GSTs from four speciesThe residue numbering refers to the D. melanogaster GST Epsilon 6. The superimposed structures are 4YH2 (the present study, D. melanogaster), 2IMI (A. gambiae), 3ZML (A. funestus) and 3VWX (M. domestica).

Mentions: Previously, we have carried out structure–function studies on Delta class GSTs, which identified an interface lock-and-key ‘clasp’ motif that is structurally conserved within the Delta class enzymes [18]. Amino acid sequence alignments predict that this motif may also be conserved in the Epsilon class GSTs. The structural motif appears to affect protein dynamics and therefore influences substrate specificity, enzyme activity and protein stability. Analysis of the clasp formation in this Epsilon DmGSTE6 reveals that the interface motif is more complex than observed for the Delta class. The Epsilon clasp motif comprises an extended ‘wafer’ arrangement of four histidines (two contributed from each subunit), which is supported by interactions with several conserved serines from helices 3, 4 and 6 (Figure 1). This elongated motif stretches across the interface deep into both subunits of the homodimer. His101 from one subunit wraps around the His101 from the other subunit to generate the clasp motif also seen in the Delta GST class [18]. This arrangement of the histidines involves aromatic ring stacking and pi–pi interaction of the two residues. In Delta class GSTs, the clasp motif has been shown to stabilize the quaternary structure as well as have a role in subunit communication between active sites [18]. The structural contributions of the clasp motif in the Delta GST also have an impact on catalytic specificity and the efficiency of the enzyme. In the Epsilon DmGSTE6, a second histidine in each subunit, His69, interacts with clasp His101 from the other subunit (Figure 1). These His–His interactions are also supported by three serines, Ser68, Ser104, Ser163, in each subunit (Figure 1). This extended motif would appear to play a structural role, as it is internal and spans the subunit interface. Furthermore, Ser68 directly interacts with GSH in the active site as the Ser68 OG and N atoms are within 2.5–3.3 Å of the GSH O11 and O12 atoms (Figure 1). The His69 ND1 atom is also within the interaction distance of 3.5 Å of the GSH O12 atom. Both Ser68 and His69 are located at the N-terminus of alpha helix 3 and possibly exploit the helix dipole to strengthen these interactions. Residues Ser68 and His69 in DmGSTE6 are the equivalent residues to Ser65 and Arg66, in the active site of the Delta class AdGSTD3, which are reported to be components of an electron-sharing network utilized for catalysis [19,27,28]. An amino acid sequence alignment of the six Epsilon GSTs, for which structures are available, is shown in Figure 2. The His wafer residues are conserved in these Epsilon class proteins and superposition of their structures shows that this sequence conservation translates into side chain position conservation in their 3D structures (Figure 3).


Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

Wongsantichon J, Robinson RC, Ketterman AJ - Biosci. Rep. (2015)

Superposition of the histidine interface motif of Epsilon GSTs from four speciesThe residue numbering refers to the D. melanogaster GST Epsilon 6. The superimposed structures are 4YH2 (the present study, D. melanogaster), 2IMI (A. gambiae), 3ZML (A. funestus) and 3VWX (M. domestica).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660579&req=5

Figure 3: Superposition of the histidine interface motif of Epsilon GSTs from four speciesThe residue numbering refers to the D. melanogaster GST Epsilon 6. The superimposed structures are 4YH2 (the present study, D. melanogaster), 2IMI (A. gambiae), 3ZML (A. funestus) and 3VWX (M. domestica).
Mentions: Previously, we have carried out structure–function studies on Delta class GSTs, which identified an interface lock-and-key ‘clasp’ motif that is structurally conserved within the Delta class enzymes [18]. Amino acid sequence alignments predict that this motif may also be conserved in the Epsilon class GSTs. The structural motif appears to affect protein dynamics and therefore influences substrate specificity, enzyme activity and protein stability. Analysis of the clasp formation in this Epsilon DmGSTE6 reveals that the interface motif is more complex than observed for the Delta class. The Epsilon clasp motif comprises an extended ‘wafer’ arrangement of four histidines (two contributed from each subunit), which is supported by interactions with several conserved serines from helices 3, 4 and 6 (Figure 1). This elongated motif stretches across the interface deep into both subunits of the homodimer. His101 from one subunit wraps around the His101 from the other subunit to generate the clasp motif also seen in the Delta GST class [18]. This arrangement of the histidines involves aromatic ring stacking and pi–pi interaction of the two residues. In Delta class GSTs, the clasp motif has been shown to stabilize the quaternary structure as well as have a role in subunit communication between active sites [18]. The structural contributions of the clasp motif in the Delta GST also have an impact on catalytic specificity and the efficiency of the enzyme. In the Epsilon DmGSTE6, a second histidine in each subunit, His69, interacts with clasp His101 from the other subunit (Figure 1). These His–His interactions are also supported by three serines, Ser68, Ser104, Ser163, in each subunit (Figure 1). This extended motif would appear to play a structural role, as it is internal and spans the subunit interface. Furthermore, Ser68 directly interacts with GSH in the active site as the Ser68 OG and N atoms are within 2.5–3.3 Å of the GSH O11 and O12 atoms (Figure 1). The His69 ND1 atom is also within the interaction distance of 3.5 Å of the GSH O12 atom. Both Ser68 and His69 are located at the N-terminus of alpha helix 3 and possibly exploit the helix dipole to strengthen these interactions. Residues Ser68 and His69 in DmGSTE6 are the equivalent residues to Ser65 and Arg66, in the active site of the Delta class AdGSTD3, which are reported to be components of an electron-sharing network utilized for catalysis [19,27,28]. An amino acid sequence alignment of the six Epsilon GSTs, for which structures are available, is shown in Figure 2. The His wafer residues are conserved in these Epsilon class proteins and superposition of their structures shows that this sequence conservation translates into side chain position conservation in their 3D structures (Figure 3).

Bottom Line: Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance.The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order.This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138673.

No MeSH data available.