title: Mass spectrometric determination of disulfide bonds in the biologically active recombinant HBx protein of hepatitis B virus.
abstract: Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of their folded state and to keep regulatory control over their functions. The hepatitis B virus-encoded HBx oncoprotein is known to perform an overwhelming array of functions in the cell and has been implicated in the development of hepatocellular carcinoma. However, its structure has not been elucidated. HBx carries nine conserved cysteine residues that have proven to be crucial for its various functions. However, the status of disulfide bonds between the cysteine residues reported in previous studies remains discrepant because of the use of refolded recombinant HBx that may contain non-native disulfides. Now we have determined the disulfide linkages in soluble and biologically active recombinant maltose binding protein-HBx fusion protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We report four disulfide linkages in HBx protein, viz., between Cys(7) and Cys(69), Cys(61) and Cys(115), Cys(78) and Cys(137), and Cys(17) and Cys(143), based on the differential mobility of corresponding disulfide-linked peptide ions under reducing and nonreducing conditions. Cys(148) was observed to be free. Site-directed mutagenesis of Cys(143) and Cys(148) with serine and functional analyses of these mutants affirmed the importance of these residues in the ability of HBx to potentiate Cdk2/cyclin E kinase activity and transcriptionally activate promoter reporter gene activity. Thus, this study identifies native disulfide linkages in the structure of a biologically active viral oncoprotein.
authors: Virology Group and ‡Plant Transformation Group, International Center for Genetic Engineering and Biotechnology , Aruna Asaf Ali Marg, New Delhi 110067, India.
authors: Kaveri
authors: K
authors: Sidhu
authors: Saravanan
authors: S
authors: Kumar
authors: Vanga Siva
authors: VS
authors: Reddy
authors: Vijay
authors: V
authors: Kumar
mesh_terms: D019927:Cyclin E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D019927:Cyclin E; D004220:Disulfides; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D003545:Cysteine