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Publication Detail
Identification of the C3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Hinshelwood J, Spencer DI, Edwards YJ, Perkins SJ
  • Publication date:
  • Pagination:
    587, 599
  • Journal:
    Journal of Molecular Biology
  • Volume:
  • Issue:
  • Print ISSN:
  • Keywords:
    Binding Sites, recombinant, homology, modelling, peptides, Peptide Fragments, Recombinant Proteins, Proteins, Amino Acid Sequence, analysis, Biochemistry, chemistry, Complement 3, Complement Factor B, genetics, metabolism, Methods, Models, Molecular, Molecular Biology, Molecular Sequence Data, Protein Conformation, Sequence Homology, Amino Acid, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Support, Non-U.S.Gov't, von Willebrand Factor, SP, control, Structure, Receptor, regulation, function, ANS
  • Notes:
    UI - 20079520 LA - Eng RN - EC (Complement Factor B) RN - 0 (von Willebrand Factor) RN - 0 (Complement 3) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Proteins) PT - JOURNAL ARTICLE DA - 20000118 IS - 0022-2836 SB - M SB - X CY - ENGLAND JC - J6V AA - Author EM - 200003
Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface- enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH3) in the presence of Mg(2+). A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central beta-sheet of the factor B vWF-A structure. The peptides G229- K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A beta-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions. Copyright 1999 Academic Press
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