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Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pK,) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases. Portrait of a superfamily of detoxification enzymesMany sophisticated defense strategies have evolved in organisms enabling them to deal with the constant threat by a broad spectrum of both foreign and endogenous cytotoxic and genotoxic compounds [1]. Enzyme systems that transact the chemical detoxification and elimination processes of these structurally diverse molecules, many of which are highly non-polar, can be divided into three major but interrelated groups; phase I enzymes, such as the cytochrome P450 superfamily, activate chemicals by forming reactive functional groups (e.g. epoxides) in them; phase I1 enzymes deactivate these reactive chemicals by appending a hydrophilic moiety (e.g. glutathionyl, glucuronyl, or sulphuryl) to the functional group ; phase Ill enzymes mediate the cellular elimination of the inactive and water-soluble products.The superfamily of glutathione S-transferases (EC 2.5.1.18) represents an integral part of the phase I1 detoxification mechanism. These intracellular proteins are found in most aerobic eukaryotes and prokaryotes, and protect cells against chemical-induced toxicity and stress by catalyzing the S-conjugation between the thiol group of glutathione and an electrophilic moiety in the hydrophobic and toxic sub- GST, glutathione S-transferase; pGSTP1-1, hGSTAl-1, rGSTM1-1 etc., acronyms for the glutathione S-transferases (GST), the prefix indicating the species (p, porcine; h, human ; r, rat; b, bovine; m, mouse; rb, rabbit; c, chicken; gp, guinea pig) while P, A, and M indicate gene class pi, alpha and mu, respectively ; 1-1 indicates a dimer of two type-1 subunits; GSH, reduced glutathione; P1, P2 etc. and G1, G2 etc., designate peptide functional groups in glutathione and the corresponding G-site ligands of the glutathione S-transferases, respectively; G-site, glutatbione-binding site; H-site, hydrophobic electrophile-binding site.Enzyme. Glutathione S-transferase (EC 2.5.1.1 8).strate. Glutathione S-transferases are perhaps the single most important family of enzymes involved in the metabolism of alkylating compounds [2]. The resultant glutathione S-conjugates can be exported from animal cells by putative membrane ATP-dependent pump systems [3] after which they are metabolized via the mercapturate pathway and eventually eliminated. An extraordinary feature of the glutathione S-transferase superfamily is the occurrence of multiple enzyme forms that, according to sequence similarities and subc...

The three‐dimensional structure of class pi glutathione S‐transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1–74) consists of a central four‐stranded beta‐sheet flanked on one side by two alpha‐helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81–207) contains five alpha‐helices. The dimeric molecule is globular with dimensions of about 55 A ×52 A ×45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S‐transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.

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The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.

A glutathione S-transferase (Sj26GST) from Schistosoma japonicurn, which functions in the parasite's Phase I1 detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea-and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea-and temperaturegradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfoldinghefolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a AG"(H20) = 26.0 k 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of AC,, = 7,440 J/mol per K, AH = 950.4 kJ/mol, and AS = 1,484 J/mol.

CLIC proteins function as anion channels when their structures convert from a soluble form to an integral membrane form. While very little is known about the mechanism of the conversion process, channel formation and activity are highly pH-dependent. In this study, the structural properties and conformational stability of CLIC1 were determined as a function of pH in the absence of membranes to improve our understanding of how its conformation changes when the protein encounters the acidic environment at the surface of a membrane. Although the global conformation and size of CLIC1 are not significantly altered by pH in the range of 5.5-8.2, equilibrium unfolding studies reveal that the protein molecule becomes destabilized at low pH, resulting in the formation of a highly populated intermediate with a solvent-exposed hydrophobic surface. Unlike the intermediates formed by many soluble pore-forming proteins for their insertion into membranes, the CLIC1 intermediate is not a molten globule. Acid-induced destabilization and partial unfolding of CLIC1 involve helix alpha1 which is the major structural element of the transmembrane region. We propose that the acidic environment encountered by CLICs at the surface of membranes primes the transmembrane region in the N-domain, thereby lowering the energy barrier for the conversion of soluble CLICs to their membrane-inserted forms.