Kinetic characterization of native and cysteine 112-modified glutathione S-transferase A1-1: reassessment of nonsubstrate ligand binding

We have prepared human glutathione S-transferase isoform A1-1 (GST A1-1) which has been chemically modified at cysteine 112. These modifications include formation of mixed disulfides with glutathione ("glutathiolation") and cross-linkage of the GST dimer with bis-maleimides reacting with the equivalent Cys 112 residues of the two monomers. This residue (Cys 112) lies adjacent to the hydrophobic substrate binding site, and its side chain thiol projects into the large, solvent-filled cleft which is widely reported in the literature to be the binding site of nonsubstrate ligands. Both types of modification block this intersubunit cleft region and significantly change its chemical environment. Kinetic experiments with these altered enzymes revealed that neither type of modification affects the catalytic activity of GST A1-1 or the binding of nonsubstrate ligands. The lack of an effect on glutathione conjugation activity is somewhat surprising given the proximity of cysteine 112 to the hydrophobic substrate binding site. More surprising, however, is the observation that modification at cysteine 112 has no effect on the binding of nonsubstrate ligands. Furthermore, two of these ligands, lithocholic acid and estradiol disulfate, unexpectedly exhibited competitive inhibition of the unmodified enzyme, suggesting that they bind in the hydrophobic substrate site rather than some accessory ligand binding site. Together, these results strongly argue against the intersubunit cleft as the nonsubstrate ligand binding site and prompt a reassessment of how these ligands interact with GST A1-1.     

Residue 219 impacts on the dynamics of the C-terminal region in glutathione transferase A1-1: implications for stability and catalytic and ligandin functions

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.     

1.3-A resolution structure of human glutathione S-transferase with S-hexyl glutathione bound reveals possible extended ligandin binding site

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.     

The hydrophobic lock-and-key intersubunit motif of glutathione transferase A1-1: implications for catalysis, ligandin function and stability

A hydrophobic lock-and-key intersubunit motif involving a phenylalanine is a major structural feature conserved at the dimer interface of classes alpha, mu and pi glutathione transferases. In order to determine the contribution of this subunit interaction towards the function and stability of human class alpha GSTA1-1, the interaction was truncated by replacing the phenylalanine 'key' Phe-51 with serine. The F51S mutant protein is dimeric with a native-like core structure indicating that Phe-51 is not essential for dimerization. The mutation impacts on catalytic and ligandin function suggesting that tertiary structural changes have occurred at/near the active and non-substrate ligand-binding sites. The active site appears to be disrupted mainly at the glutathione-binding region that is adjacent to the lock-and-key intersubunit motif. The F51S mutant displays enhanced exposure of hydrophobic surface and ligandin function. The lock-and-key motif stabilizes the quaternary structure of hGSTA1-1 at the dimer interface and the protein concentration dependence of stability indicates that the dissociation and unfolding processes of the mutant protein remain closely coupled.     

A conserved N-capping motif contributes significantly to the stabilization and dynamics of the C-terminal region of class Alpha glutathione S-transferases

Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.     

Energetics of ligand binding to human glutathione transferase A1-1: Tyr-9 associated localisation of the C-terminal helix is ligand-dependent

A C-terminal helix (α9) adjacent to the active site on each subunit is a structural feature unique to the alpha isoform of glutathione transferases which contributes to the catalytic and ligandin functions of the enzyme. The ionisation state of Tyr-9, a residue critical to catalysis, influences α9 dynamics, although the mechanism is poorly understood. In this study, isothermal titration calorimetry was used to probe the binding energetics of G-site (glutathione and glutathione sulfonate) and H-site (ethacrynic acid) ligands to wild-type and a Y9F mutant of human glutathione transferase A1-1. Although previous studies have reported a favourable entropic component to the binding of conjugates occupying both sites, our data reveal that ligand binding is enthalpically driven when either the G- or H-site is occupied independently. Also, heat capacity changes demonstrate that α9 is fully localised by H-site but not G-site occupation. The Tyr-9 hydroxyl group contributes significantly to ligand binding energetics, although the effect differs between the two binding sites. G-site binding is made slightly enthalpically more favourable and entropically less favourable by the Y9F mutation. Binding to the H-site is more dramatically affected, with the K(d) for ethacrynic acid increasing 5 fold despite a more favourable ΔS. The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔC(p)=-0.73 kJ mol(-1) K(-1)), but less negative for H-site binding to the Y9F mutant (ΔΔC(p)=0.63 kJ mol(-1) K(-1)). This suggests that the relationship between Tyr-9 and α9 is not independent of the ligand. Rather, Tyr-9 appears to function in orienting the ligand optimally for α9 closure.     

Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products.

The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal alpha-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the beta1-alpha1 loop, the end of the alpha4 helix, and the C-terminal alpha9 helix. A few residue changes in GST A4-4 result in alpha9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix alpha9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the alpha9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.     

The C-terminal region of human glutathione transferase A1-1 affects the rate of glutathione binding and the ionization of the active-site Tyr9

In human glutathione transferase (GST) A1-1, the C-terminal region covers the active site and contributes to substrate binding. This region is flexible, but upon binding of an active-site ligand, it is stabilized as an amphipatic alpha-helix. The stabilization has implications for the catalytic activity of the enzyme. In the present study, residue M208 in GST A1-1 has been mutated to Lys and Glu, and residue F220 to Ala and Thr. These mutations are likely to destabilize the C-terminal region due to loss of hydrophobic interactions with the rest of the hydrophobic binding site. The rate constant for binding of glutathione to wild-type GST A1-1 is 450 mM(-)(1) s(-)(1) at 5 degrees C and pH 7.0, which is less than for an association limited by diffusion. However, the M208 and the F220 mutations increase the apparent on-rate constant for glutathione binding to 640-1170 mM(-)(1) s(-)(1). The binding data can be explained by a rapid reversible transition between different enzyme conformations occurring prior to glutathione binding, and restriction of the access to the active site by the C-terminal region. The effect of the mutations appears to be promotion of a less closed conformation, thereby facilitating the association of glutathione and enzyme. Both the M208 and F220 mutants display a lowered pK(a) value ( approximately 0.3 log unit) of the catalytically important Tyr9. Residue 208 does not interact directly with Tyr9 in the active site, and the shift in pK(a) value is therefore ascribed to the proposed dislocation of the C-terminal region caused by the mutation.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha 9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha 9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters K(cat) and K(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha 9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on K(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.     

Molecular recognition at the dimer interface of a class mu glutathione transferase: role of a hydrophobic interaction motif in dimer stability and protein function

Cytosolic glutathione (GSH) transferases (GSTs) exist as stable homo- and heterodimers. Interactions at the subunit interface serve an important role in stabilizing the subunit tertiary structures of all GSH transferases. In addition, the dimer is required to maintain functional conformations at the active site on each subunit and the nonsubstrate ligand binding site at the dimer interface [Dirr, H. W. (2001) Chem.-Biol. Interact. 133, 19-23]. In this study, we report on the contribution of a specific intersubunit hydrophobic motif in rGSTM1-1 to dimer stability and protein function. The motif consists of the side chain of F56 from one subunit intercalated between helices 4 and 5 of the second subunit. Replacement of F56 with the hydrophilic side chains of serine, arginine, and glutamate results in a change in the structure of the active site, a marked diminution in catalytic efficiency, and alterations in the ability to bind nonsubstrate ligands. The mutations also affect the ability of the enzyme to bind GSH and the substrate analogue glutathione sulfonate. The functionality of rGSTM1-1 was disrupted to the greatest extent for the F56E mutant. Though mutations at this position do not alter the three-state equilibrium folding process for rGSTM1-1 (i.e., N(2) <--> 2I <--> 2U), destabilizing mutations at position 56 shift the equilibrium between the folded dimer (N(2)) and the monomeric intermediate (I) toward the latter conformational state. The transition to the unfolded state (U) is not significantly affected. The folded monomeric intermediate is also observed by electrospray ionization mass spectrometry. The amount of the intermediate is dependent on protein concentration and the residue at position 56. Mutations at position 56 have little impact on the secondary structure and stability of the monomeric folding intermediate. The dimerization process is proposed to induce a conformational change in the loop containing F56, resulting in improved stability and increased affinity between the M1 subunits.     

Pressure-dependent ionization of Tyr 9 in glutathione S-transferase A1-1: contribution of the C-terminal helix to a "soft" active site.

The glutathione S-transferase (GST) isozyme A1-1 contains at its active site a catalytic tyrosine, Tyr9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS-. In the substrate-free GST A1-1, the Tyr 9 has an unusually low pKa, approximately 8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan-free mutant, W21F. In addition, a short alpha-helix, residues 208-222, provides part of the GSH and hydrophobic ligand binding sites, and the helix becomes "disordered" in the absence of ligands. Here, hydrostatic pressure has been used to probe the conformational dynamics of the C-terminal helix, which are apparently linked to Tyr 9 ionization. The extent of ionization of Tyr 9 at pH 7.6 is increased dramatically at low pressures (p1/2 = 0.52 kbar), based on fluorescence titration of Tyr 9. The mutant protein W21F:Y9F exhibits no changes in tyrosine fluorescence up to 1.2 kbar; pressure specifically ionizes Tyr 9. The volume change, delta V, for the pressure-dependent ionization of Tyr 9 at pH 7.6, 19 degrees C, was -33 +/- 3 mL/mol. In contrast, N-acetyl tyrosine exhibits a delta V for deprotonation of -11 +/- 1 mL/mol, beginning from the same extent of initial ionization, pH 9.5. The pressure-dependent ionization is completely reversible for both Tyr 9 and N-acetyl tyrosine. Addition of S-methyl GSH converted the "soft" active site to a noncompressible site that exhibited negligible pressure-dependent ionization of Tyr 9 below 0.8 kbar. In addition, Phe 220 forms part of an "aromatic cluster" with Tyr 9 and Phe 10, and interactions among these residues were hypothesized to control the order of the C-terminal helix. The amino acid substitutions F220Y, F2201, and F220L afford proteins that undergo pressure-dependent ionization of Tyr 9 with delta V values of 31 +/- 2 mL/mol, 43 +/- 3 mL/mol, and 29 +/- 2 mL/mol, respectively. The p1/2 values for Tyr 9 ionization were 0.61 kbar, 0.41 kbar, and 0.46 kbar for F220Y, F220I, and F220L, respectively. Together, the results suggest that the C-terminal helix is conformationally heterogeneous in the absence of ligands. The conformations differ little in free energy, but they are significantly different in volume, and mutations at Phe 220 control the conformational distribution.     

Molecular dynamics simulations of human glutathione transferase P1-1: analysis of the induced-fit mechanism by GSH binding.

We report here a 1-ns molecular dynamics simulation on the ligand-free monomer of human glutathione transferase P1-1 in bulk water. The average conformation obtained from the last 500 ps of simulation is taken as a model for the apo-structure of this protein and compared to the available crystallographic data. Remarkable changes in the tertiary structure take place during the simulation and are ascribed to the removal of the ligand. They support an induced fit mechanism occurring upon glutathione binding, whose major features can be described in detail. A portion of helix 2 (residues 42-50), which participates in the formation of the active site, undergoes the most prominent conformational changes. Other protein segments, such as the C-terminal loop and helix 4, also show relevant structural rearrangements. All these transitions cause a significant shielding from the solvent of the hydrophobic binding site of the co-substrate, whose exposed surface goes from 4.6 nm(2) in the holo-structure to about 3.1 nm(2) in the apo-conformation. The results of this simulation are consistent with numerous experimental observations previously obtained on GST P1-1 and provide new insights for their explanation at the molecular level. Proteins 1999;37:1-9.     

Probing the conformational state of a truncated staphylococcal nuclease R using time of flight mass spectrometry with limited proteolysis.

The conformational state of C-terminally truncated staphylococcal nuclease R (SNR135), with and without bound ligands, has been studied by performing limited proteolysis with a specific endoproteinase Glu-C followed by electrophoresis and mass spectrometry. Comparison of the accessibility of the cleavage sites shows that the C-terminal truncation of 14 amino-acid residues causes significant unfolding of the C-terminal part of alpha helix 1 and the center of alpha helix 2, but there is little effect on other regions of the nuclease, in particular the N-terminal subdomain, which includes the active site of the nuclease. The truncation also makes the overall conformation of the nuclease more loose and flexible. Binding of ligands makes helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state, although the conformational stability of the SNR135 complex is still much lower than that of the full-length enzyme. The results suggest that the amino-acid residues around the active site in the truncated nuclease are arranged in a similar topology to those in the full-length nuclease. The study shows that there is a clear-cut correlation between protease susceptibility and conformational stability of the protein, and the initial proteolytic events are the most critical for evaluating the conformational features of the protein. This study demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.     

Highlighting a π–π interaction: a protein modeling and molecular dynamics simulation study on Anopheles gambiae glutathione S-transferase 1-2

Cytosolic insect theta class glutathione S-transferases (GSTs) have not been studied completely and their physiological roles are unknown. A detailed understanding of Anopheles gambiae GST (Aggst1-2) requires an accurate structure, which has not yet been determined. A high quality model structure of Aggst1-2 was constructed using homology modeling and the ligand–protein complex was obtained by the docking method. Molecular dynamics (MD) simulations were carried out to study conformational changes and to calculate binding free energy. The results of MD simulation indicate that Aggst1-2 undergoes small conformational changes after ligands dock to the protein, which facilitate the catalytic reaction. An essential hydrogen bond was found between the sulfur atom of glutathione (GSH) and the hydrogen atom of hydroxyl group in Ser9, which was in good agreement with experimental data. A π–π interaction between Phe204 and CDNB ligand was also found. This interaction seems to be important in stabilization of the ligand. Further study of binding free energy decomposition revealed a van der Waals interaction between two ligands that may play a key role in nucleophilic addition reaction. This work will be a good starting point for further determination of the biological role of cytosolic insect theta class GSTs and will aid the design of structure-based inhibitors.     

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II 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 temperature-gradient 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 unfolding/refolding 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 delta G degree (H2O) = 26.0 +/- 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 delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.     

Structural basis of urea-induced unfolding of Fasciola gigantica glutathione S-transferase

Glutathione S-transferases (GSTs) are enzymes that are involved in the detoxification of harmful electrophilic endogenous and exogenous compounds by conjugating with glutathione (GSH). The liver fluke GSTs have multifunctional roles in the host–parasite interaction, such as general detoxification and bile acid sequestration to synthase activity. The GSTs have been highlighted as vaccine candidates towards parasitic flukes. In this study, we have thoroughly examined the urea-induced unfolding of a mu-class Fasciola gigantica GST1 ( FgGST1) using spectroscopic techniques and molecular dynamic simulations. FgGST1 is a highly cooperative molecule, because during urea-induced equilibrium unfolding, a concurrent unfolding of the protein without stabilization of any folded intermediate was observed. The protein was stabilized with conformational free energy of about ~12.36 kcal/mol. The protein loses its activity with increasing urea concentration, as the GSH molecule is not able to bind to the protein. We also studied the fluorescence quenching of Trp residues and the obtained K _SV data that provided additional information on the unfolding of FgGST1. Molecular dynamic trajectories simulated in different urea concentrations and temperatures indicated that urea destabilizes FgGST1 structure by weakening hydrophobic interactions and the hydrogen bond network. We observed a precise correlation between the in vitro and in silico studies.     

Interactions of bilirubin and other ligands with ligandin.

Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.     

Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies

The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.