Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: A solution study by circular dichroism, Fourier transform infrared and H-1 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 a ctivated 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-p arallel beta-sheet flanked on both sides by seven alpha-helices A1 to A7, w ith 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 rec ombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by c ircular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg2 +. Two upfield-shifted signals in the H-1 NMR spectrum were used as sensiti ve probes of the VWF-A protein structure, one of which was assigned to a me thyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectr oscopy showed that metal-binding caused the VWF-A structure to become signi ficantly more stable. Ring current calculations based on a homology model f or 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 in dividual single protons. An allosteric property of the vWF-A domain has thu s been identified, and its implications for factor B activation were examin ed. Since the VWF-A domain after alpha-helix A7 is connected by a short lin k to the catalytic serine protease domain in the Bb fragment, the identific ation of a metal-free and a more stable metal-bound conformation for the vW F-A domain implies that the VWF-A interaction with C3b may alter its Mg2+-b ound coordination in such a way as to induce conformational changes that ma y regulate the proteolytic activity of factor B. (C) 2000 Academic Press.