Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability

Abstract

The effect of the Y108V mutation of human glutathione S-transferase P1-1 (hGST P1-1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 fi Val resulted in a 3D-structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H-site) and glutathione binding site (G-site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H-site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K d~0 .5 lM) when compared with those of the parent compounds, K EA d~1 3 lM, K GSH d~2 5 lM. The EA moiety of the conjugate binds in the H-site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the DC p values of binding can also be correlated with the potential stacking interactions between Additional Supporting Information may be found in the online version of this article.Abbreviations: DMSO, dimethylsulfoxide; DSC, differential scanning calorimetry; DTT, dithiothreitol; EA, ethacrynic acid; EACys, ethacrynic-cysteine conjugate; EAME, ethacrynic-mercaptoethanol conjugate; EASG, ethacrynic-glutathione conjugate; GST, glutathione transferase; HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; hGST P1-1, human glutathione transferase P1-1; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; MPD, 2-methyl-2,4-pentanediol; wt, wild type.Indalecio Quesada-Soriano and Lorien J. Parker contributed equally to this work.The coordinates for the structures of the Y108V apo, EA and EASG complex are deposited with the Protein Databank (http:// rcsg.org/pdb/) with the entry codes 3HJM, 3HKR, and 3HJO, respectively. ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.

Diuretic drug binding to human glutathione transferase P1‐1: potential role of Cys‐101 revealed in the double mutant C47S/Y108V

“…EASG and EACys (EA conjugate with L-cysteine) were synthesized and their purity analysed as described previously (Quesada-Soriano et al, 2009). Other chemicals employed were of the highest available purity.…”

“…The experimental conditions and data analysis were similar to those described elsewhere (Té llez-Sanz et al, 2006;Quesada-Soriano et al, 2009). The binding of active site ligands to the C47S/Y108V GST P1-1 mutant quenches the intrinsic fluorescence of the enzyme as was described for wt GST P1-1 Ortiz-Salmeró n et al, 2003;Caccuri et al, 1991).…”

“…In addition to being an inhibitor, EA can also act as a substrate, forming a conjugate with GSH via Michael addition (EASG), both spontaneously and by GST-driven catalysis, by pi, mu and alpha class GSTs (Ploemen et al, 1990;Phillips and Mantle, 1991;Awasthi et al, 1993). Recently, we investigated the role of a catalytically important residue located in the H-site, Tyr-108, in binding and catalysis of a range of GST P1-1 ligands including EA Nuccetelli et al, 1998;Quesada-Soriano et al, 2009). These studies highlighted the importance of Tyr-108: both the hydroxyl and aromatic moieties were found to be involved in the stabilization of certain ligands with changes to this residue by mutation adversely affecting ligand binding to the H-site.…”

“…In our previous work we explored methods that would allow us to prevent the covalent modification of Cys-47: by chemical modification with alkylating agents, by protection with methylSG or by modifications of the EA inhibitor itself (using EACys and EAME conjugates) (Quesada-Soriano et al, 2009). However, these procedures proved insufficient to avoid the chemical modification of the enzyme by EA and consequently prevented further investigation into the effect of Y108V mutation on the intrinsic binding of this drug to GST P1-1.…”

Thermodynamics of Molecular Recognition by Calorimetry

“…Human glutathione transferase P1-1 (hGSTP1-1), a homodimeric protein of 46 kDa, has been extensively studied for its potential use as a marker during chemical carcinogenesis and its possible role in the mechanism of cellular multidrug resistance against a number of anti-neoplastic agents (Hayes et al, 2005). S-nitroglutathione (GSNO) binds to wild-type hGSTP1-1 with negative cooperativity, whereas the C47S mutation induces positive cooperativity towards both GSNO and (ethacrynic and glutathione conjugate) EASG binding (Tellez-Sanz et al, 2006;Quesada-Soriano et al, 2009). The equilibrium between a ligand and a macromolecule with two ligand binding sites can be described in terms of two different sets of association constants: the macroscopic association constants (overall,  i , or stepwise, K i ), or the microscopic or intrinsic constants:…”