Assignment of the Four Disulfides in the N-terminal Somatomedin B Domain of Native Vitronectin Isolated from Human Plasma
Journal Article
·
· Journal of Biological Chemistry
- University of Tennessee, Knoxville (UTK)
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{sup 5}:Cys{sup 9}, Cys{sup 19}:Cys{sup 31}, Cys{sup 21}:Cys{sup 32}, and Cys{sup 25}:Cys{sup 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{sup 19}: Cys{sup 31} and Cys{sup 21}:Cys{sup 32} disulfides. A small positively charged loop is created at the N terminus by the Cys{sup 5}: Cys{sup 9} cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys{sup 25} and Cys{sup 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.
- Research Organization:
- Oak Ridge National Laboratory (ORNL)
- Sponsoring Organization:
- ORNL LDRD Seed-Money
- DOE Contract Number:
- AC05-00OR22725
- OSTI ID:
- 978053
- Journal Information:
- Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 34 Vol. 279; ISSN JBCHA3; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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