Journal of Molecular Biology
Regular articleThe structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution1
Introduction
The glutathione S-transferases (GSTs, E.C. 2.5.1.18) are a family of enzymes that protect cells against many xenobiotic substances and products of oxidative stress. They achieve this by conjugating electrophilic substrates to the tripeptide glutathione (GSH, γ-Glu-Cys-Gly) which often makes them less toxic and more readily excretable from the body. Mammalian cytosolic GSTs exist either as homo- or heterodimers with a subunit molecular mass of about 25 kDa and with one active site per monomer (for a review, see Wilce & Parker, 1994). They have been classified into at least five distinct families: alpha, mu, pi, sigma and theta based on studies of substrate specificity and primary structures Mannervik et al 1985, Meyer et al 1991, Meyer and Thomas 1995. The amino acid sequence identities between any two members within a class is typically greater than 70% whereas the figure is usually less than 30% between classes. The crystal structures of at least one representative from three of the mammalian class families have been determined: alpha (Sinning et al., 1993), mu Ji et al 1992, Raghunathan et al 1994 and pi Reinemer et al 1991, Reinemer et al 1992, Dirr et al 1994a, Garcia-Saez et al 1994. These studies show that the different isozymes adopt the same protein fold and share some similar active-site features.
The human pi-class GST (hGST P1-1) has been implicated in the development of resistance of tumors towards various anti-cancer drugs (for reviews, see Coles and Ketterer 1990, Waxman 1990, Tsuchida and Sato 1992, Hayes and Pulford 1995). A number of human tumors, including cancers of the colon, stomach, pancreas, uterine cervix, breast and lung have been shown to express raised levels of the hGST P1-1 enzyme (Coles and Ketterer 1990, Tsuchida and Sato 1992, and references therein). Tumor cell lines tend to overexpress the hGST P1-1 isozyme in particular (Montali et al., 1995). Moreover, it has been shown that selective inhibitors of hGST P1-1 potentiate the chemotherapeutic efficacy of anti-cancer drugs in resistant tumor cells (Morgan et al., 1996). Both results stress the significant potential that the design of highly potent hGST P1-1 selective inhibitors may have in increasing the therapeutic index of commonly used anti-cancer agents.
The first crystal structure of a pi-class GST, from pig lung, was determined at 2.3 Å resolution in 1991 (Reinemer et al., 1991). The structure revealed what has now turned out to be the canonical fold for all the cytosolic classes of GSTs Dirr et al 1994b, Wilce and Parker 1994. The enzyme is a homodimer with each subunit composed of two domains, as shown in Figure 1; the N-terminal (residues 1 to 74) and the C-terminal domains (residues 81 to 207). The glutathione binding site (G-site) is made up of residues mostly from the N-terminal domain whilst the binding site for electrophilic substrates (H-site) is made up of residues predominantly from the C-terminal domain. A medium-resolution (2.8 Å) structure of the human pi-class enzyme complexed with S-hexyl GSH (Reinemer et al., 1992) and high-resolution structures of the pig enzyme in complex with GSH sulfonate (Dirr et al., 1994a) and of a mouse pi-class enzyme in complex with GSH sulfonate, S-hexyl GSH and S-(p-nitrobenzyl) GSH (Garcı́a- Sáez et al., 1994) have since been published.
Here we present high-resolution structures of the human enzyme in complex with GSH, S-hexyl GSH and γ-glutamyl-(S-benzyl)cysteinyl-d-phenylglycine (TER-117). The last compound has been reported as a potent inhibitor of hGST P1-1 (Lyttle et al., 1994). As the medium-resolution structure of the human enzyme and the high-resolution structures of the pig and mouse enzymes have been analysed in detail elsewhere Reinemer et al 1992, Garcia-Saez et al 1994, Dirr et al 1994a, Dirr et al 1994b we have chosen to give only a brief overview of the high-resolution model of the enzyme and to concentrate our analysis on features of the new structures that have not been discussed previously. The data presented here provide a wealth of detail about the enzyme’s active site including information about protein flexibility, water structure and ligand-protein interactions, all of which will be of great value in designing new inhibitors that may prove useful in chemotherapy.
Section snippets
S-hexyl GSH complex
The structure of this complex was previously reported at a resolution of 2.8 Å in the space group P43212 (Reinemer et al., 1992). By changing the reducing agent from β-mercaptoethanol to DTT and the buffer from phosphate to Mes, a new crystal form has been obtained that diffracts to a much higher resolution (see Materials and Methods; Table 1). The structure of the complex determined from the new crystal form has been refined to a resolution of 2.0 Å with a crystallographic R-factor of 19.7%
Crystallization, data collection and processing
The protein was overexpressed in Escherichia coli and purified as described (Battistoni et al., 1995). Because the protein was purified by affinity chromatography using GSH as an eluant, the final protein sample had GSH bound to the G-site. Crystallization was performed by the hanging-drop vapor diffusion method (McPherson, 1982) using 24-well tissue-culture plates. A 2 μl droplet of a 8 mg/ml protein solution containing 10 mM phosphate buffer (pH 7.0), 1 mM EDTA and 2 mM mercaptoethanol, was
Acknowledgements
We thank Dr Matthew Wilce for his work on the early stages of this project. M.W.P. is an Australian Research Council Senior Research Fellow and A.J.O. was a recipient of a National Health & Medical Research Council Postgraduate Research Scholarship and an International Centre for Diffraction Data Crystallography Scholarship. We gratefully acknowledge the financial support of the Anti-Cancer Council of Victoria and the National Research Council (grant no. 96.03710.CT14) of Italy.
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Edited by R. Huber