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

¹ Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, Faculty of Experimental Sciences, University of Almería, 04120 Almería, Spain
² Department of Biology and
³ Internal Medicine, University of Rome "Tor Vergata," 00133 Rome, Italy
⁴ Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

(RECEIVED December 20, 2005; FINAL REVISION January 31, 2006; ACCEPTED January 31, 2006)

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 2 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 2 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

Reprint requests to: Luis García-Fuentes, Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, Faculty of Experimental Sciences, University of Almería, La Cañada de San Urbano, 04120 Almería, Spain; e-mail: lgarcia{at}ual.es; fax: +34-950-015008.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052055206.

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-N'-2-ethanesulfonic acid; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; PEG, polyethylene glycol.

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