title: Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.
abstract: Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K(D)(1) of 2.6 microM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences Darwin Building, University College London, Gower Street, London, UK.
authors.forename: Azubuike I
authors.initials: AI
authors.lastname: Okemefuna
authors.forename: Keying
authors.initials: K
authors.lastname: Li
authors.forename: Ruodan
authors.initials: R
authors.lastname: Nan
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Ormsby
authors.forename: Tania
authors.initials: T
authors.lastname: Sadlon
authors.forename: David L
authors.initials: DL
authors.lastname: Gordon
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perkins
mesh_terms: D015933:Complement C3d / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D017242:Complement Factor H / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation; D014961:X-Ray Diffraction
chemical_list: D015933:Complement C3d; D017242:Complement Factor H