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 Cys(5):Cys(9), Cys(19):Cys(31), Cys(21):Cys(32), and Cys(25):Cys(39). 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 Cys(19): Cys(31) and Cys(21):Cys(32) disulfides. A small positively charged loop is created at the N terminus by the Cys(5): Cys(9) cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys(25) and Cys(39) similar to cystine-stabilized alpha-helical structures commonly observed in cystine knots. This alpha-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.
Horn
Nancy A
NA
Department of Biochemistry and Cellular and Molecular Biology and the Center of Excellence in Structural Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Hurst
Gregory B
GB
Mayasundari
Anand
A
Whittemore
Neil A
NA
Serpersu
Engin H
EH
Peterson
Cynthia B
CB
D001798:Blood Proteins / Q000737:chemistry*; D004220:Disulfides; D006801:Humans; D017434:Protein Structure, Tertiary; D013002:Somatomedins / Q000737:chemistry*; D019096:Vitronectin / Q000737:chemistry*
D001798:Blood Proteins; D004220:Disulfides; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B