title: S-Nitrosation destabilizes glutathione transferase P1-1.
abstract: Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand , Johannesburg, South Africa.
authors.forename: David
authors.initials: D
authors.lastname: Balchin
authors.forename: Stoyan H
authors.initials: SH
authors.lastname: Stoychev
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D015536:Down-Regulation / Q000187:drug effects; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009566:Nitrates / Q000378:metabolism*; D015538:Nitrosation / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D058849:Protein Refolding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
chemical_list: D007202:Indicators and Reagents; D009566:Nitrates; D011994:Recombinant Proteins; D026422:S-Nitrosoglutathione; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D003545:Cysteine