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.
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.
Hinshelwood
J
J
Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, UK.
Perkins
S J
SJ
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D011415:Complement Factor B / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014841:von Willebrand Factor / Q000737:chemistry*
D008670:Metals; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012996:Solutions; D014841:von Willebrand Factor; D011415:Complement Factor B; D008274:Magnesium