A structure-based mechanism of cisplatin resistance mediated by glutathione transferase P1-1.

Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.

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

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S-transferase (GST P1-1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr-108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys-47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X-ray diffraction. Surprisingly, in the absence of Cys-47, Cys-101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys-47. The Cys-47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G-site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H-site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H-site and in the water network at the dimer interface.

Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences.

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.

Nitrosylation of human glutathione transferase P1-1 with dinitrosyl diglutathionyl iron complex in vitro and in vivo.

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.