Assignment of the four disulfides in the N-terminal somatomedin B domain of native vitronectin isolated from human plasma
The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys5:Cys9, Cys 19:Cys31, Cys21:Cys32, and Cys 25:Cys39. This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys19: Cys31 and Cys21:Cys32 disulfides. A small positively charged loop is created at the N terminus by the Cys5: Cys9 cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys25 and Cys 39 similar to cystine-stabilized α-helical structures commonly observed in cystine knots. This α-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor.
Publication Source (Journal or Book title)
Journal of Biological Chemistry
Horn, N., Hurst, G., Mayasundari, A., Whittemore, N., Serpersu, E., & Peterson, C. (2004). Assignment of the four disulfides in the N-terminal somatomedin B domain of native vitronectin isolated from human plasma. Journal of Biological Chemistry, 279 (34), 35867-35878. https://doi.org/10.1074/jbc.M405716200