Conformational changes during the assembly of factor B from its domains by (1)H 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 activated C3 (C3b or C3(H(2)O)). The Ba fragment contains three short consensus/complement repeat domains, while the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain, all three of which are implicated in multisite contacts with C3. The upfield-shifted signals in the (1)H NMR spectra of factor B, the Ba and Bb fragments, and the vWF-A and SP domains were used as sensitive conformational probes of their structures. Temperature studies and pH titrations showed that the Ba fragment 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 of factor D in the absence of substrate. The NMR spectra from the separate vWF-A and SP domains in combination with that of the Ba fragment generally accounted for that of intact factor B, apart from the perturbation of an upfield-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 vWF-A or SP domains or intact factor B. Ring current calculations based on homology models or crystal structures predicted that buried hydrophobic methyl-aromatic interactions probably accounted for the upfield-shifted signals, with many arising from the N-terminal subdomain of the SP domain to which the C terminus of the vWF-A domain is directly linked. It was concluded that: (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 which conserved charged residues may be important. Allosteric structural rearrangements in the SP domain as the result of its interactions with the vWF-A domain or the Ba fragment provide an explanation of the regulation of the catalytic activity of factor B.
Hinshelwood
J
J
Department of Biochemistry and Molecular Biology, Royal Free Campus, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, NW3 2PF, UK.
Perkins
S J
SJ
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003176:Complement C3 / Q000737:chemistry; D011415:Complement Factor B / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011416:Complement Factor D / Q000737:chemistry; D018360:Crystallography, X-Ray; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D014841:von Willebrand Factor / Q000737:chemistry
D003176:Complement C3; D010446:Peptide Fragments; D014841:von Willebrand Factor; D006859:Hydrogen; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B