3KM6

Crystal Structure of the Human GST Pi C47S/Y108V Double Mutant in Complex with the Ethacrynic Acid-Glutathione Conjugate

Summary for 3KM6

• Entry DOI: 10.2210/pdb3km6/pdb
• Related: 3KMN 3KMO
• Descriptor: Glutathione S-transferase P, ETHACRYNIC ACID, GLUTATHIONE, ... (5 entities in total)
• Functional Keywords: transferase, glutathione, detoxification, double mutant, ethacrynic acid, diuretic drug, dimer interface
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 2
• Total formula weight: 47794.32

Authors

Parker, L.J. (deposition date: 2009-11-10, release date: 2010-03-23, Last modification date: 2023-11-01)

Primary citation

Quesada-Soriano, I.,Parker, L.J.,Primavera, A.,Wielens, J.,Holien, J.K.,Casas-Solvas, J.M.,Vargas-Berenguel, A.,Aguilera, A.M.,Nuccetelli, M.,Mazzetti, A.P.,Lo Bello, M.,Parker, M.W.,Garcia-Fuentes, L.
Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V.
J.Mol.Recognit., 24:220-234,

PubMed Abstract: 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.

PubMed: 20540076
DOI: 10.1002/jmr.1040

Experimental method

X-RAY DIFFRACTION (2.1 Å)