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Comparative Study
. 2001 Nov;10(11):2176-85.
doi: 10.1110/ps.ps.21201.

The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B

A J Oakley  1 , T Harnnoi, R Udomsinprasert, K Jirajaroenrat, A J Ketterman, M C Wilce
Affiliations
Comparative Study

The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B

A J Oakley et al. Protein Sci. 2001 Nov.

Abstract

Glutathione S-transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1-3 (complexed with glutathione) and AdGST1-4 (apo form), at 1.75 and 2.45 A resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C-terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione-binding helix alpha2 and flanking residues are disordered in the AdGST1-4 (apo) structure, yet ordered in the AdGST1-3 (GSH-bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi- and human pi-class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1-4 and AdGST1-3 have different conformations.

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Figures

Fig. 1.
Fig. 1.
(a) Stereo diagram of electron density of GSH in AdGST1–3 (σa-weighted 2fo-fc map shown in gold at a contour of 1σ) with surrounding G-site residues. Electron density of H-site residues for AdGST1–4 (b) and AdGST1–3 (c) with σa-weighted 2fo-fc maps contoured at 1σ are shown in blue. Ball and stick representation is used for all atoms and bonds.
Fig. 2.
Fig. 2.
Comparison of AdGST1–3, AdGST1–4, and LcGST structures. Monomers of AdGST1–3 (a) and AdGST1–4 (b) are shown in ribbon form with the five-amino-acid insert in AdGST1–4 between the N- and C-terminal domains colored orange. The C-terminal helices (α8) of both enzymes are colored yellow. (c) Superimposed structures of AdGST1–3 (green), AdGST1–4 (blue), and LcGST monomers (orange). (d) Stereo diagram of the H-sites of AdGST1–3 (yellow) and AdGST1–4 (blue). The fold is represented in ribbon form with putative H-site amino acids shown in ball and stick form. The GSH model is of that found in AdGST1–3. Numbers correspond to residues in AdGST1–3. (e) Stereo diagram of superimposed structures of AdGST1–3 (green), AdGST1–4 (blue), and LcGST (orange) in the vicinity of helix α8. The residue W197 in Lucilia GST overlapping helix α8 in AdGST1–3 and AdGST1–4 is indicated in ball-and-stick form. (f) Comparison of AdGST 1–3 (yellow) versus AdGST1–4 (blue) with helices shown as cylinders. Helix α8 and the region around and including helix α2 is highlighted in orange (for AdGST1–3) and cyan (for AdGST1–4). The shifts in AdGST1–4 relative to AdGST1–3 are indicated.
Fig. 3.
Fig. 3.
Structure-based sequence alignment of insect GSTs. Secondary structure is indicated above the sequences in red. The kink in helix α4 is indicated (Θ). The hydrophobic staple at the base of helix α6 is indicated (<>), as is the N-terminal capping m ([]). Regions of high structural conservation are boxed with amino acids indicated in bold upper case. Residue numbers are those for AdGST1–4. The region of AdGST1–4 with no electron density (around helix α2) is indicated with a horizontal line. Helix α8, unique to the AdGST isozymes is indicated. The helix observed linking the N- and C-terminal domains of AdGST1–4 is indicated as α3B. G-site residues have shaded backgrounds. H-site residues have white letters on a black background.
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