Complement Factor H: solution structures and interactions with ligands.
Doctoral thesis, UCL (University College London).
Factor H (FH) is a plasma glycoprotein that plays a central role in regulating the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and host cell-surface polyanionic ligands. C3d is a 35-kDa fragment of C3b, the activated form of the central complement protein C3. Creactive protein (CRP) is an acute-phase reactant that activates complement through the classical pathway but inhibits the alternative pathway. In this thesis, X-ray scattering, analytical ultracentrifugation (AUC) and constrained modelling were used to determine solution structures for the SCR-1/5 and SCR-16/20 fragments, as well as for intact FH. Surface plasmon resonance (SPR) was used in conjunction with these methods to investigate the interactions of FH with C3d and CRP in the fluid and solid phases. Structural studies revealed that at physiological concentrations, SCR-1/5 is monomeric and SCR-16/20 exists in a weak monomer-dimer equilibrium. In the best-fit models, both FH fragments adopt a partially folded-back orientation in solution, and the SCR-16/20 dimer is formed by association of two SCR-20 domains. FH exists in a partially folded-back orientation in solution and forms oligomers. Here, these FH oligomers were shown to exist under different buffer conditions of NaCl concentration and pH, and these two factors also influence the conformation of FH. New, significantly improved molecular structures showed that FH orientation is maintained by short-to-middle distance interdomain interactions. There was no evidence of long distance interactions between the N- and C-terminals of FH. Studies on the interaction of FH with C3d identified a number of multimeric complexes formed between C3d and both SCR-16/20 and native FH. Binding to C3d did not significantly increase the overall length of FH, and this interaction is NaCl concentration-dependent. CRP, another ligand of FH, is known to exist physiologically as a pentamer. Here, CRP is shown to exist in a pentamer-decamer equilibrium under physiological buffer conditions. This equilibrium is NaCl concentration dependent and occurs in both fluid and solid phases. SPR binding studies showed that interaction with CRP inhibits FH oligomerisation, and further identified a novel CRP-binding site within SCR-16/20 of FH. Thus ionic strengthdependence may be a general feature of FH interaction with its ligands. These results provide insight into the complement regulatory activity of FH.
|Title:||Complement Factor H: solution structures and interactions with ligands|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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