Calorimetric and structural studies of the nitric oxide carrier S‐nitrosoglutathione bound to human glutathione transferase P1‐1

Abstract

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix a2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix a2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.Keywords: calorimetry; glutathione S-transferase; nitric oxide; nitrosoglutathione; X-ray crystallography Supplemental material: see www.proteinscience.org Glutathione transferases (EC 2.5.1.18; GSTs) are a superfamily of enzymes involved in the biotransformation of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds (Jakoby and Habig 1980). The human cytosolic GSTs are dimeric proteins grouped into at least eight species-independent classes (a, k, m, p, v, s, u, and z) on the basis of their amino acid sequence, substrate specificity, and immunological properties (Mannervik et al. 1985;Meyer et al. 1991;Buetler and Eaton Reprint requests to: Luis García-Fuentes, Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, Faculty of Experimental Sciences, University of Almería, La Cañ ada de San Urbano, 04120 Almería, Spain; e-mail: lgarcia@ual.es; fax: +34-950-015008.Abbreviations: DSC, differential scanning calorimetry; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; GST, glutathione S-transferase; hGSTP1-1, human glutathione transferase P1-1; HEPES, N-(2-hydroxyethyl)-piperazine-N9-2-ethanesulfonic acid; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; PEG, polyethylene glycol.Article published online ahead of print. Article and publication date are at

Redox compartmentalization in eukaryotic cells

“…Albumin also contains a redox-active Cys residue which is normally about 50% present as the S-cysteinyl disulfide form. This thiol has been suggested to function as a carrier of NO and also to function in detoxification [146,147]. The content of this disulfide was also found to correlate with the plasma Cys/CySS redox potential [138].…”

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

“…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).…”

“…The sample preparation and ITC experiments were carried out as previously described elsewhere (Té llez-Sanz et al, 2006;Quesada-Soriano et al, 2009). Titrations were routinely performed in 20 mM sodium phosphate, 5 mM NaCl, 0.1 mM EDTA at pH 7.…”

Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1

“…Recently, the crystal structure of S-nitrosoglutathione bound to hGSTP1-1 showed that the S-nitrosoglutathione ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the enzyme. 23 The crystallographic analysis also revealed three possible conformations of the nitroso group, which could be modeled with ~33% occupancy for each. In one conformation the nitrosyl moiety was stabilized by water-mediated interactions with Arg13 and Tyr108.…”