Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer

Abstract

To determine the location of the non-substrate-ligand-binding region in mammalian glutathione Stransferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-a glutathione S-transferase, and in a human Trp284Phe mutant class-n glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-a and class-n enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligandprotein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis Mol. Biol. 246,. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.Keywords: glutathione S-transferase; energy transfer; non-substrate ligand; 8-anilinonaphthalene 1 -sulfonate.Cytosolic glutathione S-transferases (GST) a superfamily of heterodimeric and homodimeric proteins, catalyse the nucleophilic addition of the thiol of reduced glutathione (y-glutamylcysteinylglycine) to electrophilic centres in a wide variety of hydrophobic electrophiles, including alkyl and aryl halides, epoxides, quinones and activated alkenes (Armstrong, 1991). Although they can be grouped into five species-independent gene classes ( a, p, z, r~ and O), GST share a common protein fold (Dirr et al., 1994a). Three-dimensional structures of isoenzymes from the a (Sinning et al., 1993;Cameron et al., 1995), p (Ji et al., 1992Raghunathan et al., 1994), n (Reinemer et al., 1992; Dim et al., 1994b; Garcia-Saez et al., 1994), r~ (Ji et al., 1995) and B (Wilce et al., 1995; Reinemer et al., 1996) classes have been determined, and this abundance of structural information has provided much insight into the protein-architectural relationships between the different gene classes. Each glutathione Stransferase subunit is folded into two distinct structural domains. Domain 1 has an papappa folding topology, in which its conserved core provides the principal framework for the active site. Domain 2 has an all-a-type core structure. There are two structurally independent active siteddimer, each of which consists of a glutathione-binding site (G-site) and an electrophilic-hydrophobic-substrate-binding site (H-site).

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

“…The fluorescenceresonance-energy-transfer measurements suggest that the aflatoxin B1 binding site is located at the dimer interface cleft close to the crystallographic two-fold axis. This is the same site at which the anionic dye ANS is bound [20]. Competitive ligand binding studies confirm this result.…”

“…4 A). This site was recently identified as the ANS binding site in human GST A1-1 [20]. Double-reciprocal plots of ANS binding to human GST A1-1 in the presence of aflatoxin B1 (data not shown) confirms that both of these ligands compete for the same binding site.…”

“…In the crystal structures of the human GST A1-1 [23,24], the thiol groups of the cysteine residues face in toward the large V-shaped cleft formed at the dimer interface. ANS binds in this V-shaped cleft [20] and it is logical to assume that alkylation of the cysteine residue with a bulky reagent (such as IAEDANS) would block the cleft and impede ANS from binding.…”

“…Ϫ14 [20]). The quantum yield of the donor fluorophore (Trp20) is 0.057 (see [20] for the calculation and discussion of this value].…”

“…The quantum yield of the donor fluorophore (Trp20) is 0.057 (see [20] for the calculation and discussion of this value]. If both donor (Trp20) and acceptor (aflatoxin B1) molecules rotate freely in a time that is short compared with the excited-state lifetime of the donor, the orientation factor (K 2 ) assumes a value of 2/3 [21].…”