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Complement is an essential component of the innate and acquired immune system1, and consists of a series of proteolytic cascades that are initiated by the presence of micro-organisms. In health, activation of complement is precisely controlled through membrane-bound and soluble plasma-regulatory proteins including factor H (fH)2, a 155 kDa protein composed of twenty domains (termed complement control protein repeats, or CCPs). Many pathogens have evolved the ability to avoid immune- killing by recruiting host complement regulators3 and several pathogens have adapted to avoid complement-mediated killing by sequestering fH to their surface4. Here we present the first structure of a complement regulator in complex with its pathogen surface-protein ligand. This reveals how the important human pathogen Neisseria meningitidis subverts immune responses by mimicking the host, using protein instead of charged-carbohydrate chemistry to recruit the host complement regulator, factor H. The structure also indicates the molecular basis of the host-specificity of the interaction between factor H and the meningococcus, and informs attempts to develop novel therapeutics and vaccines.
Neisseria meningitidis is a human adapted pathogen of global importance as a leading cause of bacterial meningitis and septic shock5. Due to Neisserial strain variation, the vaccines currently available for meningococcal disease are only effective against subsets of strains and do not provide universal protection6,7. However, promising novel antigens have been identified8, including factor H binding protein (known as fHbp, GNA1870, or R2086), a 27 kDa surface lipoprotein which is present on the surface of all strains of N. meningitidis and elicits protective bactericidal antibodies9,10. The structure of a portion of fHbp has been determined by NMR11, and its function studied by subdividing it into a series of regions, termed “A”, “B” and “C” 12. fHbp is the sole receptor for fH on the meningococcus, and recruitment of fH contributes to the ability of the meningococcus to avoid innate immune responses by inhibiting complement-mediated lysis in human plasma13,14. Individuals with polymorphisms in the promoter of the gene encoding fH that are associated with increased plasma fH levels are at increased risk from meningococcal disease15.
We have used a range of approaches to dissect the fH binding sites on fHbp (Fig. 1). High affinity interactions between fH and fHbp were only observed with fHbp constructs containing all three of the previously defined (see above) regions of fHbp (Fig. 1a), implying that fHbp has an extended recognition site for fH across its entire surface. We then sought to identify which of the 20 CCP domains of fH mediate the interaction with fHbp. Far Western, FACS (Fig. 1b) and Surface Plasmon Resonance (Fig. 1c) analyses demonstrated that the key regions of fH recognised by fHbp are the sixth and seventh domains, CCPs 6 & 7 (known hereafter as fH67) and that a construct containing both these domains was capable of inhibiting the fHbp-dependent interaction between fH and N. meningitidis (Fig. 1d). Quantification of the interaction demonstrates that the dissociation constant is ~5nM (Fig. 1c & 1e, Tab. S1, Fig. S1) for any construct containing CCPs 6 & 7. This interaction was not dissociated by high salt (1M NaCl, not shown) or pH ranging from 4 to 8 (not shown), providing additional support for the high affinity nature of the binding event.
To better understand fH:fHbp recognition, we crystallised and determined crystal structures of the complex between fH67 and an fHbp construct comprising regions A, B and C but lacking the lipid anchor. Models for fHbp and fH67 were built and refined to 2.35 Å in two crystal forms with a total of seven independent copies of the complex (Fig. 2). The structure reveals that fHbp folds to form two ß–barrels (Fig. 2a) with the N-terminal barrel consisting of the “A” and part of the “B” regions, whilst the C-terminal barrel (as previously seen in the NMR structure of the “BC” region11, overlaid in Fig. S2) is composed of the rest of the “B” and the “C” regions. Searching structural databases with the N-terminal barrel reveals no close structural homologues (Supplemental Analyses), and the distinct topologies of the ß-barrels (Fig. 2c) suggest that they have not arisen by a gene duplication event.
The fH67:fHbp complex is held together by extensive interactions between both ß-barrels of fHbp and fH CCP6 (Fig. 2b), with more minor contacts to CCP7, consistent with our binding studies. In particular, the helical insertion into the second ß-strand of CCP6 (an unusual feature of this CCP domain) is centrally located in the complex. Analysis by the PISA server gives all seven independent complexes a significance score of 1.0 (i.e. extremely likely to be biologically significant) with the average surface area of fHbp buried in the complex (2860±177Å2) greater than that buried in most antibody:antigen complexes16. Furthermore, the ΔG predicted on the basis of the structure for the formation of the complex (−6 kcal/mol) is in good agreement with the affinity derived from binding studies (−11 kcal/mol, calculated from KD presented in Fig. 1e), providing additional support for the physiological relevance of the crystallised complex. The interaction surface shows good shape complementarity with numerous electrostatic interactions (Fig. 2c, Fig. S3) including multiple hydrogen bonds and salt bridges. To further probe the interaction surface we generated doubly mutated forms of both proteins with charged side chains being replaced by the small hydrophobic residue Ala (fH67R341A,H337A and fHbpE283A,E304A). Using SPR to study the interactions between the mutated proteins and their wild-type partners revealed that the affinity of both mutant forms was reduced by more than two orders of magnitude so that almost no interaction could be seen at analyte concentrations (~50 nM) around ten times the wild-type KD (Fig. 3a). At a thousand-fold higher analyte concentration (μM range) the mutant forms of both proteins did interact with the wild-type partners: fHbpE283A,E304A retained a qualitatively similar on-rate but an increased off-rate with respect to wild-type, whilst fH67R341A,H337A was more similar to the wild-type interaction in its off-rate but had a vastly reduced on-rate (Tab. S1). It is interesting to note that E304 is not conserved in all Neisserial strains with a Thr also found at this position10. Since these strains also sequester fH it is likely that either the loss of a single interaction (rather than the two lost in the mutant) does not decrease the affinity sufficiently to preclude functional fH sequestration or that subtle rearrangements of the local side chains allow the Thr side chain to interact with residues in fH. The ability of the interaction to tolerate some variation in the residues at the interface would clearly be immunologically advantageous.
A common fH polymorphism (fH 402His/Tyr), recently shown to be a major susceptibility factor for age related macular degeneration17-19, lies at the periphery of the interaction site and the side chain of residue 402 (His in this structure) is within hydrogen bonding distance of residues in fHbp (Fig. S4 & S5). However, our interaction studies do not detect any differences in binding (Fig. 1e, Tab. S1) between the fH 402His/Tyr isoforms. This lack of functional consequence suggests, as for variation at the 304 position (see above) that either the loss of a single hydrogen bond from this extensive interaction surface is not sufficient to produce a functional effect or that the small amount of flexibility at the CCP 6/7 junction seen between the crystallographically independent copies of the complex allows subtle rearrangement of the fH CCP domains to enable both polymorphic variants to hydrogen bond to fHbp (Fig. S4).
Mapping published epitopes of fHbp that elicit bactericidal antibodies11,12 onto the structure demonstrates that none of the sites characterised to date lie directly in the fH recognition site. Instead, epitopes recognised by antibodies that affect fH binding20 are located around the edge of the recognition site and so are likely to inhibit fH binding by steric hindrance (Fig. S5).
Our earlier structure21 of CCPs 6, 7 & 8 of fH in complex with sucrose octasulphate (SOS), a highly sulphated analogue of glycosaminoglycans (GAGs), revealed a charged groove on fH that forms an extended interaction site for binding of GAGs on the surface of endothelial cells. This, combined with an additional GAG-binding site in CCPs 19-2022, provides the mechanism by which fH localises to mammalian cells23 to prevent inappropriate complement activation. Examination of the fHbp:fH67 complex demonstrates that the Neisserial protein binding site lies within this extended GAG interaction site, overlapping the CCP6 SOS-binding site (Fig. 3b) with no fH rearrangement upon binding fHbp (Fig S6). There are several examples of pathogens imitating their host by expressing directly related molecules (e.g. sialic acid or glycolipids24). Here, however, N. meningitidis mimics the mechanism by which host cells regulate complement activation on their surface using amino acid side-chains instead of charged sugars. Since the binding sites of fHbp and SOS on fH are overlapping, we tested whether SOS can act as a competitive inhibitor of the interaction between fH and fHbp. Strikingly, SOS prevented the fH/fHbp interaction (assessed by SPR Fig. 3d, and by FACS Fig. S7) at millimolar concentrations, supporting the relevance of the crystal structure to complex formation in solution and providing a lead compound for development of small molecule inhibitors of fH:fHbp interactions.
N. meningitidis has evolved to avoid innate responses at sites of colonisation and during disease. It is also host-restricted, with the human nasopharynx its only natural reservoir5. This host specificity can limit the significance of animal models for studying certain aspects of pathogenesis and immunity. It has previously been shown that N. meningitidis only binds human fH and not fH from other primates or other animals25. The amino acid sequences of fH from human and other primates are highly conserved (~90% identity) whilst other animal fHs are more sequence divergent. It is therefore of note that even the limited number of differences between human and primate sequence changes within CCP6 cluster to the precise site of interaction with fHbp. For instance, mapping the differences between human and rhesus macaque fH26 shows that all six differences within CCP6 lie in or around the interface (Fig. 3e). This indicates the likely structural basis for the species specificity of complement evasion25 by N. meningitidis through fH recruitment, and suggests that specific modification of fH could provide a novel approach to develop more biologically relevant models of meningococcal disease.
Interestingly, the high affinity interaction between fH and fHbp suggests that N. meningitidis could rapidly sequester fH in the plasma, depleting the circulating levels and de-regulating complement, rendering host cells in the vascular compartment more susceptible to complement-mediated damage. This might contribute to the dramatic haemorrhagic rash seen in meningococcal sepsis, and suggests that inhibitors specifically designed to block the interaction between CCP6 and fHbp might both promote complement-mediated clearance of bacteria and prevent sequestration of fH and consequent vascular damage27.
The success of meningococcal vaccines containing fHbp which are currently being evaluated may be hampered by binding of fH which, due to high affinity and the extensive sites of interaction, could cloak critical, conserved epitopes on the antigen. Structural analysis of this complex paves the way for the development of engineered fHbp vaccines in which fH binding is abolished to unveil the full array of protective epitopes including those that could generate responses to inhibit recruitment of fH by this important human pathogen.
Full methods are described in the supplementary material. Recombinant proteins were expressed and purified using affinity columns or size exclusion chromatography and protein concentration estimated using absorbance at λ=280nm. Binding assays (FACS, far Western and SPR) were performed according to published protocols. The complex was crystallised by vapour diffusion and X-ray diffraction data collected at the European Synchrotron Radiation Facility (ESRF, France) and DIAMOND (UK). The structure was solved by a combination of molecular replacement for the fH67 portion21 (1/3 of the structure) and isomorphous replacement/anomalous dispersion methods, with seven independent copies refined to 2.35Å in two different crystal forms.
Supplementary Information accompanies the paper on www.nature.com/nature.
Thanks are due to Gérard Bricogne and the Global Phasing Consortium for access to a β version of autoSHARP and autoBUSTER, to the beamline staff of the European Synchrotron Radiation Source (particularly Dr. G. Leonard) and Diamond and to many members of the Laboratory of Molecular Biophysics, Oxford and the Lea Group for help with X-ray data collection. B.E.P. was funded by a Wellcome Trust studentship P.R. & S.J. by Medical Research Council grants to S.M.L., J.E.D. by a Wellcome Trust grant to SM.L. and J.E.L. by an Engineering and Physical Sciences Research Grant to S.M.L. Work in C.M.T.'s laboratory is funded by the Wellcome Trust and the Medical Research Council. E.K. is an EMBO fellow. B.E.P, J.E.D. & J.J.E.C. performed SPR experiments, B.E.P., J.J.E.C., J.E.L., S.Q expressed proteins and crystallised the complex, M.C.S., E.K & Q.Z. performed all assays with Neisseria and expressed fHbp, S.J., P.R. and S.M.L. collected and analysed X ray data, S.M.L. phased the X-ray data and built/refined the models, S.M.L. and C.M.T. designed the research and wrote the paper.
Coordinates and X-ray data have been deposited in the protein databank with accession identifiers 2w80 & 2w81.