Conformational changes during the assembly of factor B from its domains byH-1 NMR spectroscopy and molecular modelling: Their relevance to the regulation of factor B activity

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments in the presence of activa ted C3 (C3b or C3(H2O)). The Ba fragment contains three short consensus/com plement repeat domains, while the Bb fragment contains a iron Willebrand fa ctor type A (vWF-A) domain and a serine protease (Sr) domain, all three of which are implicated in multisite contacts with C3. The upfield-shifted sig nals in the H-1 NMR spectra of factor B, the Ba and Bb fragments, and the V WF-A and SP domains were used as sensitive conformational probes of their s tructures. Temperature studies and pH titrations showed that the Ba fragmen t and the VWF-A and SP domains had conformationally mobile structures. The comparison of the NMR spectra of the SP domains of both factor B and factor D showed that the factor D linewidths were broader than those for factor B , which may result from a range of proteolytically inactive conformations o f factor D in the absence of substrate. The NMR spectra from the separate V WF-A and SP domains in combination with that of the Ba fragment generally a ccounted for that of intact factor B, apart from the perturbation of an upf ield-shifted signal from the Ba fragment. A new upfield-shifted signal was observed in the Bb fragment that was not detected in the spectra for the VW F-A or SP domains or intact factor B. Ring current calculations based on ho mology models or crystal structures predicted that buried hydrophobic methy l-aromatic interactions probably accounted for the upfield-shifted signals, with many arising from the N-terminal subdomain of the Sr domain to which the C terminus of the VWF-A domain is directly linked. It was concluded tha t: (1) the conformation of the free SP domain is better ordered in solution than that of factor D; (2) the conformation of the Ba fragment is affected by its incorporation into factor B; and (3) the proximity of the vWF-A and SP domains within the Bb fragment leads to a conformational change in whic h conserved charged residues may be important. Allosteric structural rearra ngements in the Sr domain as the result of its interactions with the VWF-A domain or the Ba fragment provide an explanation of the regulation of the c atalytic activity of factor B. (C) 2000 Academic Press.