Figure 1.
Structure and immunogenicity of V1 fHbps with impaired fH binding.
(A) Structures of V1 fHbp mutants with reduced binding. Top left shows overlay of cartoon representation of fHbp structures from V1 fHbp (grey), fHbpDM (gold), and fHbpR106A (teal). Zoom boxes show close-ups of modified residues with different densities (FO-FC) contoured at 4 sigma. Inset panels show typical equilibrium fits for binding to fH67, and average KD and quality of fit indicators based on four repeats. Binding of V1 fHbp and modified fHbps to fH67 by SPR (B), and by Coomassie straining (blue bands) or far Western analysis with fH (black bands) to these proteins and lysates from N. meningitidis strain MC58 and MC58ΔfHbp, the fHbp mutant (C). (D) Antibody titres against V1 fHbps elicited by immunisation with fHbps.
Table 1.
Effect of mutations at positions equivalent to fHbp V1 residues 283 and 304 (i.e. Thr304 in V2 and V3 fHbp) on the KD for binding to fH67, shown relative to the wild-type proteins.
Figure 2.
Key residues in fHbp necessary for high affinity interactions with fH.
Amino acids of V1, V2 and V3 fHbps (A, B and C respectively, fill representations) superimposed on the structure of V1 fHbp with fH (black sticks) demonstrating the impact of residues on the KD as a percentage of results with the corresponding wild-type protein. Substitution of residues coloured yellow increases binding by > fivefold, while amino acids labelled in orange have a significant effect on binding proteins from all three variant groups.
Figure 3.
Structures and stability of V2 and V3 fHbps.
(A) Overlay of V1 and V3 fHbp:fH67 complexes shown in a cartoon representation with the V1 complex shown in light grey, and V3 fHbp (rainbow coloured blue at the N-terminus to red at the C-terminus) with fH67 from the V3 complex shown in black. (B) Overlay of V3 fHbp (stick representation) in the region of residue 106 (magenta) and V3 fHbpP106A (carbon-green, oxygen-red, nitrogen-blue). (C) Overlay of V3 fHbp (rainbow coloured blue to red, N- to C-terminus) with the C terminal barrel of V2 fHbp (grey); pictures drawn by PyMol. (D) DSC of V1 (red line), V2 (blue line) and V3 (green line) fHbp showing unfolding of the C-terminal barrel at around 80°C for all variants, and the N-terminal barrel at lower temperatures.
Figure 4.
Structural basis for the reduced affinity of mfH with fHbp.
(A) Cartoon of hfH67 viewed from through V1 fHbp (solid line) with amino acids changed in hfH with murine residues (outlined by yellow dashes), and those replaced in mfH with human residues (outlined by light blue dashes). (B) SPR analysis of binding of two hfH67 mutants each containing two amino acid changes (shown) with fHbps from each variant family. (C) Far western analysis of V1 fHbp and a control protein, PPX; blots were overlaid with 5 µg/ml of the recombinant proteins mfH, modified mfH (with 14 humanised amino acids) or hfH, or with human serum (1 in 2000 dilution) as indicated; the sizes of the mol. wt. marker are shown. (D) Structure of mfH67 (blue ribbon) superimposed on V1 fHbp (white ribbon) and hfH (green ribbon). While fH6 from both species are superimposable, the orientation of fH7 differs significantly between mfH and hfH (indicated in red dashed circle).
Figure 5.
Non-functional fHbps retain their immunogenicity in transgenic mice.
(A) ELISAs assaying anti-V1 titres elicited in pooled sera following immunisation of transgenic mice with the wild-type protein and non-functional V1 fHbps. (B) SBA titres of sera from individual mice immunisation with fHbps.