title: Membrane accessibility of glutathione.
abstract: Regulation of the ion pumping activity of the Na+,K+-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane was found. Therefore, the most likely mechanism whereby the cysteine residue could become glutathionylated is via a loosening of the α-β subunit association, creating a hydrophilic passageway between them to allow access of glutathione to the cysteine residue. By such a mechanism, glutathionylation of the protein would be expected to anchor the modified cysteine residue in a hydrophilic environment, inhibiting further motion of the β-subunit during the enzyme's catalytic cycle and suppressing enzymatic activity, as has been experimentally observed. The results obtained, therefore, suggest a possible structural mechanism of how the Na+,K+-ATPase could be regulated by glutathione.
authors.affiliation: School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Garcia
authors.affiliation: School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Nasma D
authors.initials: ND
authors.lastname: Eljack
authors.affiliation: School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Marc-Antoine
authors.initials: MA
authors.lastname: Sani
authors.affiliation: School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Frances
authors.initials: F
authors.lastname: Separovic
authors.affiliation: Department of Cardiology, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia; Kolling Institute, University of Sydney, Sydney, New South Wales 2065, Australia.
authors.forename: Helge H
authors.initials: HH
authors.lastname: Rasmussen
authors.affiliation: Center for BioMembrane Physics, University of Southern Denmark, Odense M5230, Denmark.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kopec
authors.affiliation: Center for BioMembrane Physics, University of Southern Denmark, Odense M5230, Denmark.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Khandelia
authors.affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Flemming
authors.initials: F
authors.lastname: Cornelius
authors.affiliation: School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Clarke
mesh_terms: D005978:Glutathione / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008956:Models, Chemical; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D008051:Lipid Bilayers; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione
keywords: Beta-subunit; Glutathionylation; Phosphatidylcholine; Plasma membrane; Regulation; Sodium pump