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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

Hinshelwood, J; Spencer, DIR; Edwards, YJK; Perkins, SJ; (1999) 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. J MOL BIOL , 294 (2) 587 - 599.

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Abstract

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 CS(NH,) in the presence of Mg2+. 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 CS(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 vWFA 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. (C) 1999 Academic Press.

Type: Article
Title: 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
Keywords: complement C3, factor B, vWF-A domain, SELDIAMS, homology modelling, VON-WILLEBRAND-FACTOR, PLATELET GLYCOPROTEIN IB, CRYSTAL-STRUCTURE, SECONDARY STRUCTURE, PROTEIN STRUCTURES, CATION-BINDING, INTEGRIN, ACTIVATION, FRAGMENT, PREDICTIONS
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/119650
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