Structural evidence for Arabidopsis glutathione transferase At GSTF2 functioning as a transporter of small organic ligands

Abstract

Glutathione transferases (GSTs) are involved in many processes in plant biochemistry, with their best characterised role being the detoxification of xenobiotics through their conjugation with glutathione. GSTs have also been implicated in noncatalytic roles, including the binding and transport of small heterocyclic ligands such as indole hormones, phytoalexins and flavonoids. Although evidence for ligand binding and transport has been obtained using gene deletions and ligand binding studies on purified GSTs, there has been no structural evidence for the binding of relevant ligands in noncatalytic sites. Here we provide evidence of noncatalytic ligand-binding sites in the phi class GST from the model plant Arabidopsis thaliana, AtGSTF2, revealed by X-ray crystallography. Complexes of the AtGSTF2 dimer were obtained with indole-3-aldehyde, camalexin, the flavonoid quercetrin and its non-rhamnosylated analogue quercetin, at resolutions of 2.00, 2.77, 2.25 and 2.38 Å respectively. Two symmetry-equivalent-binding sites (L1) were identified at the periphery of the dimer, and one more (L2) at the dimer interface. In the complexes, indole-3-aldehyde and quercetrin were found at both L1 and L2 sites, but camalexin was found only at the L1 sites and quercetin only at the L2 site. Ligand binding at each site appeared to be largely determined through hydrophobic interactions. The crystallographic studies support previous conclusions made on ligand binding in noncatalytic sites by AtGSTF2 based on isothermal calorimetry experiments (Dixon et al. (2011) Biochem J438, 63-70) and suggest a mode of ligand binding in GSTs commensurate with a possible role in ligand transport.

Keywords: Arabidopsis thaliana; AtGSTF2; flavonoids; glutathione transferase; ligand transport; structural biology.

Figure 1

Ligands Used in this Study. 1 = Indole‐3‐aldehyde; 2 = Camalexin; 3 = Quercetrin; 4 = Quercetin.

Figure 2

Structure of AtGSTF2 dimer. The figure is derived using the complex with indole‐3‐aldehyde and shows selected helices and location of ligand‐binding sites L1 and L2 labelled for ease of reference.

Figure 3

Structure of dimers ‘A/B’ from ligand complex structures of AtGSTF2 and showing location of ligands in binding sites L1 and L2. I: Complex with Indole‐3‐aldehyde 1; II: Complex with Camalexin 2; III: Complex with Quercetrin 3; IV; Complex with Quercetin 4; V: 1GNW, an AtGSTF2 complex with two molecules of S‐hexyl glutathione ‘GSX’, showing the GSH conjugation site 30.

Figure 4

Electrostatic surface views of AtGSTF2. (A) Same view as Fig. 3 V, in complex with two molecules of S‐hexyl glutathione (PDB code 1GNW 30); (B) In complex with quercetrin 3, rotated 90°, and revealing ligand‐binding site L1; (C) In complex with quercetrin 3, rotated 180°, and revealing ligand‐binding site L2.

Figure 5

(A) Binding of indole‐3‐aldehyde 1 in the L1 site. (B) Binding of indole‐3‐aldehyde 1 in the L2 site. Backbone and side chains of monomers A and B of a dimer of AtGSTF2 are shown in ribbon and cylinder format in blue and gold respectively. Indole‐3‐aldehyde 1 is shown in ball‐and‐stick format with the carbon atoms in grey. Electron density map is shown in blue and corresponds to the F _o ‐F _c omit map contoured at a level of 3σ, which was obtained from refinement prior to the building of the ligand(s). Ligand atoms from the ligand complex structures have been added afterwards for clarity. Selected distances, given in Ångstroms, between protein and ligand atoms are indicated as bold dashed lines.

Figure 6

Binding of camalexin 2 in the L1 site. Backbone and side chains of monomers A and B of a dimer of AtGSTF2 are shown in ribbon and cylinder format in blue and gold respectively. Camalexin 2 is shown in ball‐and‐stick format with the carbon atoms in grey. Electron density map is shown in blue and corresponds to the F _o ‐F _c omit map contoured at a level of 3σ, which was obtained from refinement prior to the building of the ligand(s). Ligand atoms from the ligand complex structures have been added afterwards for clarity.

Figure 7

(A) Binding of quercetrin 3 in the L1 site. (B) Binding of quercetrin 3 in the L2 site. Backbone and side chains of monomers A and B of a dimer of AtGSTF2 are shown in ribbon and cylinder format in blue and gold respectively. Quercetrin 3 is shown in ball‐and‐stick format with the carbon atoms in grey. Electron density map is shown in blue and corresponds to the F _o ‐F _c omit contoured at a level of 3σ, which was obtained from refinement prior to the building of the ligand(s). Ligand atoms from the ligand complex structures have been added afterwards for clarity. Selected interactions between protein and ligand are indicated as bold dashed lines with distances given in Ångstroms.