Determination of the disulfide bond arrangement of Newcastle disease virushemagglutinin neuraminidase - Correlation with a beta-sheet propeller structural fold predicted for Paramyxoviridae attachment proteins

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Unti l this study, the disulfide linkages of this HN and structurally similar at tachment proteins of other members of the paramyxoviridae family were undef ined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of New castle disease virus, isolated by reverse phase high performance liquid chr omatography, and analyzed by mass spectrometry. Analysis of peptides contai ning a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(19 6) linkages and that HN ectodomains dimerize via Cys(123), Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cy s(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subseq uent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cy s(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentiall y with peptide-N-glycosidase F and trypsin. Further treatment of this pepti de by one round of manual Edman degradation or tandem mass spectrometry est ablished Cys(344)-Cys(461) and Cys(455) Cys(465) linkages, These data, esta blishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.