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PubMed · 10756110

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.

J Hinshelwood·S J Perkins
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Abstract

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.

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Allosteric RegulationAmino Acid MotifsAmino Acid SequenceBinding SitesCircular DichroismComplement Factor BHumansHydrogen-Ion ConcentrationMagnesiumMetalsModels, MolecularMolecular Sequence DataNuclear Magnetic Resonance, BiomolecularPeptide FragmentsProtein DenaturationProtein Structure, SecondaryProtein Structure, TertiaryRecombinant ProteinsSequence AlignmentSolutionsSpectroscopy, Fourier Transform InfraredStructure-Activity RelationshipThermodynamicsvon Willebrand FactorJournal ArticleResearch Support, Non-U.S. Gov't

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BibTeXRIS

J Hinshelwood, S J Perkins. 2000-04-21. 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.. https://doi.org/10.1006/jmbi.2000.3632

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