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Publication Detail
Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Hinshelwood J, Perkins SJ
  • Publication date:
  • Pagination:
    135, 147
  • Journal:
    Journal of Molecular Biology
  • Volume:
  • Issue:
  • Print ISSN:
  • Keywords:
    0 (Metals), 0 (Peptide Fragments), 0 (Recombinant Proteins), 0 (Solutions), 0 (von Willebrand Factor), 7439-95-4 (Magnesium), ABSENCE, ACTIVATION, Allosteric Regulation, Amino Acid Motifs, Amino Acid Sequence, As, Binding Sites, Biochemistry, biology, catalytic, chemistry, Circular Dichroism, Complement Factor B, COMPONENT, domain, drug effects, EC (Complement Factor B), Form, Fourier Transform, Hydrogen-Ion Concentration, IDENTIFICATION, Infrared, Infrared Spectroscopy, interaction, link, Magnesium, May, metabolism, METAL, Metals, model, modelling, Models, Molecular, Molecular, Molecular Biology, Molecular Sequence Data, NMR, Nuclear Magnetic Resonance, Biomolecular, Other, PATHWAY, Peptide Fragments, pharmacology, PROBE, Protease, PROTEIN, Protein Denaturation, Protein Structure, Protein Structure, Secondary, Protein Structure, Tertiary, Protons, recombinant, Recombinant Proteins, sensitive, Sequence Alignment, Serine, signals, SITE, Solutions, SPECTRA, spectroscopy, Spectroscopy, Fourier Transform Infrared, SPECTRUM, Structure, Structure-Activity Relationship, Support, Non-U.S.Gov't, Temperature, Thermodynamics, transition, UK, von Willebrand Factor
  • Addresses:
    Department of Biochemistry and Molecular Biology, --- *** Royal Free and University College Medical School, University College London, Rowland Hill Street, London, UK
  • Notes:
Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments when complexed with the activated form of C3, namely C3b. The Bb fragment contains a von Willebrand factor type A (vWF-A) domain, which is composed of an open twisted almost-parallel beta-sheet flanked on both sides by seven alpha-helices A1 to A7, with a metal coordination site at its active-site cleft. Homology modelling of this vWF-A domain shows that the metal-binding site was present. Two recombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by circular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg(2+). Two upfield-shifted signals in the (1)H NMR spectrum were used as sensitive probes of the vWF-A protein structure, one of which was assigned to a methyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectroscopy showed that metal-binding caused the vWF-A structure to become significantly more stable. Ring current calculations based on a homology model for the vWF-A structure correlated one upfield-shifted signal with a methyl group on the alpha-helices in the vWF-A structure and the other one with individual single protons. An allosteric property of the vWF-A domain has thus been identified, and its implications for factor B activation were examined. Since the vWF-A domain after alpha-helix A7 is connected by a short link to the catalytic serine protease domain in the Bb fragment, the identification of a metal-free and a more stable metal-bound conformation for the vWF-A domain implies that the vWF-A interaction with C3b may alter its Mg(2+)-bound coordination in such a way as to induce conformational changes that may regulate the proteolytic activity of factor B. Copyright 2000 Academic Press
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