Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function

Negative cooperativity in enzyme reactions—in which the first event makes subsequent events less favorable—is sometimes well understood at the molecular level, but its physiological role has obscure. Negative cooperativity occurs in human glutathione transferase (GST) when binds the family and its were we studied 16 different GSTs, revealing negative cooperativity is

the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (though smaller values of nH are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role -safeguarding DNA from DNDGICdisplay a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs beside the well-known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.
Glutathione transferases are a superfamily of enzymes responsible for the metabolism and inactivation of a broad range of carcinogens and xenobiotics (1,2). They catalyze the conjugation of glutathione (GSH) to many toxic organic compounds provided with an electrophilic center. They are also able to act as ligandins, binding and sequestering many types of toxins without any chemical reaction involved. This non-catalytic role has an important physiological impact because these proteins are abundantly expressed in all organisms, from bacteria to humans, approaching concentrations close to 1 mM in some cells (3). Cytosolic GSTs have been grouped into 13 gene-independent classes, based upon their primary structure (4). An alternative classification is possible on the basis of the residue that in the active site (the G-site) favors the activation of GSH: the Cys-GSTs (Omega and Beta classes) whose structures are close to the ancestral precursor of all GSTs, the Ser-GSTs (Delta, Theta, Zeta and Phi classes, including also Nu-GST activated by a threonine), and the Tyr-GSTs (Alpha, Pi, Mu and Sigma classes); this latter sub-family comprises the more recently evolved GSTs. Most of these variants are dimeric proteins composed of identical subunits. However, heterodimeric GSTs composed of different members from the same class also occur (5). For many years GSTs were considered non-cooperative enzymes based on hyperbolic binding curves of substrates (6), and further supported by the kinetic independence of subunits in heterodimeric GSTs demonstrated with various substrates as well as inhibitors (7,8). However, in 1995 the replacement of a critical cysteine (Cys47) by site-directed mutagenesis of the human GSTP1-1 disclosed a hidden inter-subunit communication, made evident by a marked positive homotropic behavior for GSH binding (9). A few years later, other single point mutations on the same variant (10)(11)(12) as well as on other GSTs (13)(14) also revealed hidden cooperativity. Inter-subunit communication in native GSTP1-1 was indicated by modification with thiol reagents (15), and the crystal structure (16) provided strong evidence for negative cooperativity in the enzyme. A number of different chemical or physical inactivators provided further support for this interpretation (17). In the meantime, a few other GSTs revealed cooperative properties in a native status, namely the murine GSTA4-4 (18), the human GSTA1-1 (19) and the Plasmodium falciparum GST (20,21). However, the human GSTP1-1 remained the most peculiar and striking case of cooperativity among all GSTs, showing positive cooperativity for GSH binding at temperatures above 35 °C and negative cooperativity below 25 °C (22). A particularly interesting GST ligand is the dinitrosyl-diglutathionyl-iron-complex (DNDGIC) (Fig. 1A) formed spontaneously when an excess of nitric oxide is produced in the cell (23). By sequestering free iron and NO, this complex may counteract both oxidative and nitrosylative stress. However, DNDGIC is toxic to the cell as indicated by the significant induction of the SOS DNA repair systems (24), activation of the oxidative shock response (soxA) gene, and the gene Sfia inhibiting cell division (25). Moreover, it irreversibly inactivates glutathione reductase, an enzyme necessary for maintaining the redox balance of the cell (26). A few GST variants have been found to act as efficient DNDGIC ligandin proteins, displaying astonishing affinities. In particular, the recently evolved Tyr-GSTs show K D values ranging from 10 -9 to 10 -12 M (23) making this complex a prime intracellular ligand for these enzymes. X-ray diffraction data demonstrated that DNDGIC binds to the G-site with loss of one glutathione (Fig. 1B), replacing it with the residue contributing to the GSH activation, i.e. tyrosine, serine or cysteine that complete the coordination shell of the ferrous ion (27). By testing 39 different GSTs, we also found that the Cys-GSTs, which are close to the ancestral GST protein have a thousand times lower affinity for DNDGIC than the Tyr-GSTs and Ser-GSTs (23). Thus, protection of the cell against NO by means of GSTs was proposed to recently have been acquired during evolution. Interestingly, Cys-GSTs are the only known GSTs expressed by bacteria, and their low affinity for DNDGIC has been related to the sensitivity of these organisms to nitrosylative stress (23). Notwithstanding the beneficial effects of the DNDGIC sequestration by GSTs, a likely disadvantage is that these enzymes, when saturated reversibly by this complex, become catalytically inactive and lose their ability to conjugate GSH to many other toxins. How is it possible to reconcile these two opposite needs, i.e. bind DNDGIC too tightly enough to be not easily released, and at the same time to preserve an essential and sufficient conjugating activity? The development of negative cooperativity for DNDGIC binding in GSTs appears to be an advantageous solution. This paper shows that negative cooperativity is not restricted to GSTP1-1, but is present also in many other members of the GST superfamily never described as cooperative enzymes. We propose that negative cooperativity has evolved to maintain a residual conjugating activity of GST against toxins in the presence of DNDGIC. By this mechanism the physiological role of detoxication is safeguarded by a phenomenon, which may be reasonably defined as "cooperative self-preservation of function".

DNDGIC binding triggers a strong negative cooperativity in many GSTs -
In the present study 16 different GSTs, as representative members of the Tyr-, Serand Cys-GST sub-families, were examined in relation to their interaction with DNDGIC. Data reported in Table 1 show that almost all tested Tyr-GSTs and Ser-GSTs display a noticeable negative cooperativity for DNDGIC binding with Hill coefficients, n H, ranging from 0.51 to 0.75 (Table 1). If the adjacent subunit is still catalytically active in the half-saturated GSTs, this behavior would warrant a residual conjugating activity even in the presence of large amount of DNDGIC, i.e. in case of relevant NO production in the cell. Notably, the Hill coefficients found for a few Tyr-GSTs approach the value of 0.5, which is the highest degree of negative cooperativity commonly observed in a dimeric enzyme (28). None of the GSTs taken as representative members of the Cys-GSTs subfamily shows any cooperativity (Table 1 and Fig. 2). Thus, these GSTs, all close to the ancestral precursor of GSTs, not only display three orders of magnitude lower affinity for DNDGIC than the Tyr-and Ser-GSTs, but also lack of any detectable inter-subunit communication necessary for cooperative modulation. Interestingly, the average of both n H and the ratio of the two apparent binding constants K D2 /K D1 for DNDGIC binding appear to be higher for the more recently evolved Tyr-GSTs than in the Ser-GSTs (Fig.  2), following a trend which parallels the evolution pathway of these enzymes. Representative fits of experimental data to the equations diagnostic for cooperativity are reported in Figs. 3, 4 and 5.
The negative cooperativity preserves the enzymatic activity in the adjacent subunit -By evaluating the effect of the cooperative binding of DNDGIC on the classical GST activity, i.e. its ability to conjugate GSH to 1chloro-2,4-dinitrobenzene (CDNB), it is evident that many tested GSTs belonging to the Tyr-and Ser-subfamilies efficiently protect the activity of the adjacent subunit after the first one has bound DNDGIC. This may be quantified by means of a "protection factor" (K i2 /K i1 -1) obtained by kinetic data, which indicates to what extent one subunit becomes more resistant to inactivation by DNDGIC when the adjacent subunit has bound this compound (Fig. 6). Among all tested Tyr-GST variants, GSTP1-1 and O. volvulus GST show the highest protection factors, but even the human GSTM2-2 and GSTA2-2 display remarkable protection values (Fig. 6). Among the Ser-GST variants, GSTD3 and GSTD5 are those showing the most efficient self-protection, while this mechanism is absent in L. cuprina GST (Table 2). Obviously, no protection is obtained in the Cys-GSTs, which do not display any cooperativity ( Fig. 6 and Table 1 and 2). Two apparently paradoxical exceptions have been found, represented by GSTA1-1 and GSTA3-3 (Table 2), and their behavior will be explored more deeply below. Excluding these two variants, the average of K i2 /K i1 ratios produce similar values as calculated from the average of K D2 /K D1 ratios (coming from thermodynamic fluorescence data) for all the Tyr-GSTs and the Ser-GSTs (Table 1 and 2). This coincidence occurs because binding of DNDGIC to one GST subunit leaves intact the catalytic efficiency of the adjacent subunit, as suggested by a residual activity in the half-saturated GSTs of about 50% of the original one. Actually, in the DNDGIC-half-saturated enzyme the affinity for GSH in the vacant subunit is not much changed. For example the half-saturated GSTP1-1 shows a K m = 0.22 mM for GSH instead of 0.15 mM (data not shown). This increment does not modify the catalytic capacity of GSTs in vivo given that the GSH concentration in the cells is 1-8 mM. Figure 7 explains the cooperative self-preservation mechanism found in GSTs. Even in the presence of DNDGIC in concentrations essentially saturating one subunit, the second subunit remains unliganded and catalytically active.
A few Alpha class GSTs adopt a half-of-thesites-interaction. -An interesting exception to the above described self-preservation found in the Tyr-GSTs and Ser-GSTs, is represented by the Alpha class GSTA1-1 and GSTA3-3, both following a typical half-of-the sites interaction, i.e. when one subunit binds DNDGIC, the adjacent one becomes fully inactive but still able to bind a second DNDGIC molecule with lower affinity (see Table 1 and 2 and Fig. 6). This property, observed in the past for GSTA1-1 (29), is now found also for GSTA3-3, while the homologous GSTA2-2 follows the cooperative self-preservation behavior demonstrated for other GSTs. This finding is proved by plotting the residual activity of these GST as a function of sub-stoichiometric additions of the inhibitor (Figs. 8A, B and C). The peculiar affinities of these two variants towards DNDGIC and their expression and localization inside the cell suggest some possible comments. In fact, GSTA1-1 and GSTA3-3 display the highest affinity, showing twenty times higher affinity for DNDGIC (about 8 ⋅ 10 -11 M) than all other Mu and Pi variants (1.5 ⋅ 10 -9 M) (23). The complete loss of activity following the halfof-the-sites binding of DNDGIC seems to be the price that needs to be paid by these GSTs for assuming the role of interceptors towards DNDGIC. This suicide behavior inherent in their enzymatic activity is probably well tolerated by the cell as the Alpha class GSTs are often co-expressed with a conspicuous level of Mu class GSTs, which show cooperative self-preservation and then guarantee a residual detoxifying activity towards other toxins in case of NO insults. Another safeguard is the presence of heterodimers containing a GSTA2 subunit, which could display activity when the other subunit is binding DNDGIC. Graphical representation of the expression of all GSTs in human hepatocyte, their detoxifying capacity toward DNDGIC and their conjugating activity towards toxic electrophilic compounds is given in Fig. 9A, B, and D. It appears that all the defense capacity against DNDGIC in human hepatocytes is exclusively assumed by the Alpha GSTs, in particular by GSTA1-1 ( Fig  9C). The human GSTA3-3 is primarily expressed in steroidogenic tissues, but not in liver (30), thus its contribution in human hepatocytes to bind DNDGIC is null despite its strong affinity for this complex (Fig 9).
A further important element which justifies the sacrifice of catalytic activity in the Alpha GSTs in favor of an acquired extraordinary affinity for DNDGIC is the peculiar cell localization of these variants. In fact, about 30% of the entire pool of Alpha-GSTs is concentrated near the inner and outer nuclear envelope, forming, as with many other enzymes, a defense barrier for DNA termed "nuclear shield" (31,32). For this peculiar role, some Alpha GSTs developed an extraordinary affinity during evolution, but, as it appears, at the expense of their catalytic efficiency.
Structural requirements for negative cooperativity in GSTs -A classical structural explanation for negative or positive cooperativity is that one subunit, once it has bound a specific ligand, modifies the structure of the adjacent free subunit. The subunit interfaces, as they appear from the X-ray structures of many dimeric GSTs, help us to identify the structural requirements for the observed negative cooperativity. The two adjacent monomers display three types of interactions: polar contacts, hydrogen bonds and hydrophobic interactions. For the mammalian Tyr-GSTs the combination of mutational, kinetic, and structural studies provide strong evidence for the structural basis of cooperativity, in particular for GSTP1-1 (33). By analogy, but with less experimental evidence, we suggest similar structural requirements for negative cooperativity in other GSTs below. In the Tyr-GSTs a typical hydrophobic contact, important for inter-subunit communication, is the "lock-and-key" motif. This structural trait is formed by an aromatic residue (key residue) from domain I in one subunit wedged into a hydrophobic pocket formed by helices 4 and 5 in domain II of the other subunit (lock apparatus) (Fig. 10A). The lock-and-key motif is a common feature of Pi, Mu, and Alpha class GSTs where the key residue is either phenylalanine or tyrosine (Tyr49 or Tyr50 with Met1 residue in hGSTP1-1) buried in a hydrophobic pocket formed by Met91, Val92, Gly95, Pro128, Phe129, and Leu132 of the second subunit chain (Fig.  10A). Mutagenesis has been used to investigate the importance of the key residue for dimerization, stability and cooperativity found in GSTP1-1 (34), GSTA1-1 (35), and GSTM1-1 (36). The S. hematobium GST, which also shows a relevant negative cooperativity (n H = 0.65) lacks this specific motif but shows an alternative insertion of an aromatic residue (Tyr92) into a hydrophobic cavity of the adjacent subunit formed by Lys80 and Met85 but also surrounded by the Arg76 and Tyr77 (Fig. 10B). In the Ser-GSTs, a lock-and-key motif is always present, but different from that reported in mammalian Alpha/Mu/Pi classes. A striking characteristic of this motif involving the 'key' residue is that it not only inserts into a hydrophobic pocket of the neighboring subunit, but also itself acts as part of the "lock" for the other subunit "key". In addition, the "key" residues from both subunits hook around each other in an aromatic pi-pi interaction, through slightly offset aromatic ring stacking, generating a "clasp" in the middle of the subunit interface (37). The "clasp" motif is formed by an aromatic residue of Phe104 of one subunit and the Arg67, His100, Leu103, and Val107 of the second subunit and vice versa occur among the Phe104 of the second subunit and the same four residues of the first subunit. The "clasp" motif appears like two hands interlocked with the two Phe104 residues in the central portion (Fig. 10C). The human GSTT2-2 lacks this peculiar motif but, similar to the S. hematobium GST, an alternative aromatic residue (Tyr73) is inserted in a hydrophobic cavity of the adjacent subunit formed by Leu89 and Ala93 possibly acting as an ancillary transmission device for cooperativity (Fig. 10D). In the Cys-GSTs no similar inter-subunit connections are observed, in full agreement with the absence of any kinetic or binding cooperativity (Table  1). Obviously, the alternative motifs proposed here for S. hematobium GST and for GSTT2-2 are plausible but only hypothetical structural mechanisms that must be confirmed in the future by mutagenesis experiments.

DISCUSSION
Negative cooperativity is even now one of the most intriguing phenomena in biochemistry but many aspects are still to be explored (38)(39)(40)(41). A typical case of negative cooperativity is the half-of-the-sites interaction, i.e. when binding of a specific inhibitor to one subunit of a dimeric enzyme results in the adjacent vacant subunit becoming fully inactive (42). Alternatively, the binding of the inhibitor to the first subunit may give the second subunit lower affinity for the same compound. In this case the adjacent subunit retains its original activity even in the presence of an excess of inhibitor (43). In this complex scenario of different and sometimes opposite effects triggered by negative cooperativity mechanisms, a much debated question is the identification of specific physiological functions. Cornish-Bowden stated in 1975 that physiological role of negative cooperativity remained unclear (44), and almost 40 years later he concluded that there had been very little progress in understanding its biological function, although he proposed that negative cooperativity can better be understood in a network context, generating very high sensitivity of metabolite concentrations to flux perturbations (45). The particular cooperativity found in the GST superfamily and described in this paper shows some novelties in this context. The interaction of GSTs with DNDGIC, a toxic compound that can be neutralized by these enzymes, but which also represents a strong inhibitor for all GSTs, offers a diverse scenario of utilization of negative cooperativity that spans from a classical half-of-the sites interaction to a cooperative self-preservation. The availability of many purified GST variants and the corresponding X-ray diffraction data offered us an extraordinary investigative strength to define evolutionary pathways and structural requirements for this phenomenon. A first conclusion is that these detoxifying enzymes have evolved to acquire a cooperative selfpreservation mechanism that helps to save a residual enzymatic activity in the case of DNDGIC overproduction i.e. after NO toxicity. The evolution progress appears evident observing that all tested Cys-GSTs (the oldest variants close to the ancestral GST), despite their dimeric structures and their three-dimensional shapes similar to the more recently evolved GSTs, display no (or very scarce) trace of negative cooperativity. In addition, most Ser-GSTs, which also appeared after Cys-GSTs but before the Tyr-GSTs in the evolution pathway of GSTs, display a less efficient cooperative selfpreservation than that found in the more recently evolved Tyr-GSTs (Table 1 and 2 and Fig. 2). A structural explanation of these differences has been recognized. The typical lock-and-key motif involving one hydrophobic residue of one subunit which is inserted between helix 4 and helix 5 of the second one, is only present in the Tyr-GSTs (35) and appears to be the most efficient structural machinery to achieve an optimized self-preservation with n H values near 0.5, which indicates a very strong negative cooperativity for a dimeric protein (see Table  1). A different but less efficient inter-subunit connection, termed "clasp" motif has been found in a few Ser-GSTs (37), but not in all. In addition, these connections are not unique: in one Tyr-GST and in one Ser-GST variants other alternative modalities of communication may be proposed (see Fig. 10B, D). Thus it appears that diverse structural motifs have been adopted by GSTs during evolution to reach an efficient inter-subunit communication. Conversely, none of the tested bacterial Cys-GSTs, close to the ancestral forms of GSTs, display any similar structural insertions. Interestingly, one of the most efficiently self-protecting GSTs is the GSTP1-1, whose enzymatic activity must definitely be saved as it is the sole GST variant expressed in some tissues. The halfof-the-sites interaction leading to full inactivation, found for the two GSTs showing the highest propensity to bind DNDGIC, is probably the price to be paid to reach such astonishing affinity. The finding that GSTs with highest affinity (i.e., the Alpha class variants) surround in large amounts the nuclear envelope is strong evidence that one primary function of these enzymes is to protect the nucleus and DNA from DNDGIC, leaving to the Mu class GSTs the classic catalytic role against other toxins. We must remember that in all cells Alpha class GSTs are often combined with variable amounts of Mu class GSTs and then this sacrifice is probably well balanced by the presence of a sufficient potential of active enzyme for the classical conjugation of GSH to other toxins (see Fig 9). Alternatively, the presence of substantial amounts of GSTA1-2 or GSTA2-3 heterodimers (5) could also guarantee a residual conjugating activity toward electrophilic toxins in the presence of DNDGIC. In fact, this appears as a reasonable explanation for the existence of GST heterodimers.
In conclusion, the cooperative selfpreservation found in GSTs, but probably also operative in a number of other multimeric enzymes, may be considered a novel biological application of Le Chatelier's principle: "If there is a change in the condition of a system in equilibrium, the system will adjust itself in such a way as to counteract, as far as possible, the effect of that change" (46). In our case, whenever a chemical factor (for example DNDGIC) perturbs or inhibits the enzyme, it opposes this perturbation by developing some negative cooperativity which tends to relieve that stress.
Other enzymes have acquired cooperative self-preservation -Many studies describing the interaction of specific inhibitors to homo-multimeric enzymes have revealed that biphasic inhibition patterns are not an unusual finding. This suggests that negative cooperativity mechanisms could have been adopted and perfected to achieve self-preservation, but curiously this possibility has only rarely been taken into consideration. An extended examination of the scientific literature now leads to the realization that hundreds of enzymes display this behavior. For example, aminoglycoside Nacetyltransferase (47), adenylosuccinate lyase (48), adenylosuccinate synthetase (49), enoyl-ACP reductase (50), thymidylate synthase (51), pyruvate kinase (43), formate dehydrogenase (52), and pyruvate phosphate dikinase (53) show negative cooperativity toward specific inhibitors but no allusion to a possible self-preservation function has been made. Conversely, the study of the prolidase inhibition by the natural inhibitor phosphoenolpyruvate represents a rare example of a discovered negative cooperativity explicitly indicated as "an expedient evolutionary solution to the problem of eluding an endogeneous inhibitor" (54).
Our intention in this paper has been to draw attention and analyze a role for negative cooperativity that has been given almost no attention in the past. However, we do not suggest that self-preservation is the only reason why it should have been selected in evolution. In other cases increasing the sensitivity of the concentrations of intermediate metabolites (45) may provide a better explanation, though it does not seem to apply to the glutathione transferases.
Synthesis of dinitrosyl-diglutathionyl iron complex -DNDGIC was prepared essentially as described previously (55). Briefly, 1 ml of 0.5 mM FeSO 4 (dissolved in degassed water to avoid rapid oxidation to the ferric state) was added to 10 ml (final volume) of 0.1 mM potassium phosphate buffer, pH 7.4, containing 20 mM GSH and 2 mM GSNO (25 °C). After 10 min, the reaction was almost complete, and the resulting stock solution of DNDGIC (50 μM) was stable for at least 3 h. More concentrated solutions (up to 1 mM DNDGIC) were also obtained by incubating, GSH 20 mM, 4 mM GSNO, and variable FeSO 4 (up to 1 mM) in 0.05 M sodium borate buffer, pH 11.0.

Inhibition by DNDGIC -The interaction of the inhibitor DNDGIC with Tyr-GSTs and
Ser-GSTs has been studied by means of inhibition experiments using the classical enzymatic reaction, i.e. GSH (0.1 mM), 1chloro-2,4-dinitrobenzene (1 mM) in 0.1 M potassium phosphate buffer and variable additions of DNDGIC. The spectrophotometric procedure was identical to the one described previously (23). Inhibition kinetics data were fitted by a two site inhibition equation 1:  Eq.2 where Y is the percent of fluorescence quenching and B max1 + B max2 = 100. As shown in Table 1  where Y is the percent of inhibition or fluorescence perturbation and n H is the Hill coefficient. Visualization of negative cooperativity (or its absence) was also obtained by means of the Hill plot (Equation 4): where Y is percent of inhibition or percent of fluorescence quenching.
Structural studies -Illustrations of X-ray structures were created by UCSF Chimera (56). PDB ID used to identify interface connectivities were: hGSTP1- Statistical data analysis -Kinetic and thermodynamic data were analyzed and displayed by GraphPad Prism software (La Jolla, CA). The experimental values reported in Table 1 and Table 2 and in Figures 3-7 are the means of three independent experiments ± SD. The propagation of uncertainties for the quotients in Table 1  Crystal structure of dinitrosyl-glutathionyl iron complex (DNGIC) bound to GSTP1-1 (one GSH is replaced by a Tyr residue which completes the coordination shell of the iron ion with its -OH group) The structure is from PDB id: 1ZGN, the protein is in green ribbons while DNGIC and Tyr7 are depicted in ball-and-stick colored according to atom type (27).

Figure 2.
Negative cooperativity for DNDGIC binding. A: Average (and standard error of the mean) of the ratio between the two dissociation constants for GST-DNDGIC interaction (K D2 /K D1 ) calculated for all tested dimeric GSTs (see Table 1) and grouped into the three sub-families. B: Average (and standard error of the mean) of all Hill coefficients calculated for all GSTs grouped into the three sub-families. Experimental details are reported under "Experimental procedures".    . Protection factors against inactivation due to DNDGIC binding. Protection factor (by what factor does one subunit become more resistant to inactivation by DNDGIC when the adjacent subunit has bound this complex) is defined as (K i2 /K i1 -1). The negative value (-1) for GSTA1-1 and GSTA3-3 (amplified in the plot) underlines an opposite phenomenon, i.e. a half-site inhibition where K i2 approaches 0. Error bars are derived from Table 2.    The lock-and-key motif in hGSTP1-1. Tyr49 (Tyr50 with Met1 residue) is reported in corn blue for chain A and in orange for chain B. Met91, Val92, Gly95, Pro128, Phe129 and Leu132 in chain A and B are reported as blue and red spheres, respectively. B: The "alternative" motif found in GST from S. haematobium. Tyr92 of the two subunits are reported in corn blue for chain A and in orange for chain B; Arg76, Tyr77, Lys80 and Met85 in chain A and B are reported as blue and red spheres respectively. C: The "clasp" motif found in GSTD4-4 from A. dirus. Phe104 of the two subunits are reported in corn blue for chain A and in orange for chain B; Arg67, His100, Leu103 and Val107 in chain A and B are reported as blue and red spheres respectively. D: The "alternative" motif of the hGSTT2-2. Tyr73 of the two subunits are reported in corn blue for chain A and in orange for chain B; Leu89 and Ala93 in chain A and B are reported as blue and red spheres respectively. In all structures the backbone chains are in blue and red colors.