PROTEIN STRUCTURE AND FOLDING
Determination of the Disulfide Bond Arrangement of Newcastle Disease Virus Hemagglutinin Neuraminidase: CORRELATION WITH A β-SHEET PROPELLER STRUCTURAL FOLD PREDICTED FOR Paramyxoviridae ATTACHMENT PROTEINS*210

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Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys531-Cys542 and Cys172-Cys196 linkages and that HN ectodomains dimerize via Cys123. Another peptide, with a chain containing Cys186 linked to a chain containing Cys238, Cys247, and Cys251, was cleaved at Met249 with cyanogen bromide. Subsequent tandem mass spectrometry established Cys186-Cys247 and Cys238-Cys251 linkages. A glycopeptide with a chain containing Cys344 linked to a chain containing Cys455, Cys461, and Cys465 was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys344-Cys461 and Cys455-Cys465 linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that theparamyxoviridae HN forms a β-propeller structural fold.

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This work was presented in part at the XIth International Congress of Virology, Sydney, Australia, August 9–13, 1999.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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The on-line version of this article (available at http://www.jbc.org) contains supplemental material including mass spectra presented in Figs. S1–S6.