Neisseria meningitidis recruits factor H using protein ... · Supplemental Methods for Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates Muriel

Supplemental Methods for

Neisseria meningitidis recruits factor H using protein

mimicry of host carbohydrates

Muriel C. Schneider1+*

, Beverly E. Prosser2*

, Joseph J.E. Caesar2, Elisabeth

Kugelberg1, Su Li

1, Qian Zhang

1, Sadik Quoraishi

2, Janet E. Lovett

2, Janet E. Deane

2,

Robert B. Sim3, Pietro Roversi

2, Steven Johnson

2, Christoph M. Tang

1 & Susan M.

Lea2

1Centre for Molecular Microbiology and Infection, Imperial College, London SW7

2AZ, U.K. 2Sir William Dunn School of Pathology, University of Oxford, South Parks

Road, Oxford OX1 3RE, U.K. 3MRC Immunochemistry Unit, Department of

Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

*These authors contributed equally to this work

Supplemental Analysis

Structure comparisons: As noted in the main text, when this structure of fHbp is compared

to the earlier structure of the “BC” region of the same protein1 there are significant

differences, particularly in the “B” region (Fig. S2). We interpret these differences as being

genuine differences between the structures due to inappropriate truncation (part way through

the N-terminal β-barrel) in the construct used for the NMR structure, rather than reflecting

differences arising from the different methods of structure determination. By contrast, when

the fH67 structure is compared to the equivalent portion from our earlier fH678/SOS complex2

(Fig. S6), no significant structural differences are seen, presumably reflecting the fact that

truncation of CCP8 does not have adverse effects on the structures of CCPs 6 and 7. No

structural homologue of the novel structure seen in the N-terminal β-barrel of fHbp is found

searching using either DALI3 or SSM 4. If more distant similarities are searched for (SSM

lower limit of acceptable secondary structure matching 40%) using the entire fHbp structure

(and ignoring topological considerations), an intriguing similarity in the arrangement of the two

β-barrels compared to the earlier structure of an engineered avidin5 (PDBID: 2C4I, Fig S10) is

observed. Given this protein’s role in complement related biology, we also note that the serine

protease domain of complement regulator Factor B6 (PDBID: 2OK5) is, in part, a two β-barrel

structure, and the relative arrangement of the two barrels is rather similar in both fHbp and

SUPPLEMENTARY INFORMATION

doi: 10.1038/nature07769

www.nature.com/nature 1

Factor B (Fig. S11). Any biological relevance of these structural similarities is, however,

unclear.

doi: 10.1038/nature07769 SUPPLEMENTARY INFORMATION


Table 1: Binding constants determined using Surface Plasmon Resonance with fHbp on the

surface of the chip and fH constructs in the fluid phase. Fits made using BiaEvaluation 2.0

software and a 1:1 Langmuir model. Values shown are mean ± standard deviation, Chi2 are

the quality of fit indicators given within BiaEvaluation 2.0. Where the sequence at residue 402

is not specified the Histidine form is used.

Construct ka (Ms-1

) kd (s-1

) KD (M) χχχχ2

fH567 3.5e5 ± 2.2e5 1.8e-3 ± 3.6e-4 6.1e-9 ± 2.5e-9 2.1 ± 1.1

fH678H402 3.6e5 ± 4.4e4 1.9e-3 ± 3.4e-4 5.7e-9 ± 2.3e-9 3.5 ± 2.2

fH678Y402 3.0e5 ± 1.9e5 1.7e-3 ± 2.8e-4 7.2e-9 ± 2.7e-9 4.3 ± 1.0

fH67 7.2e5 ± 1.5e5 2.9e-3 ± 6.4e-4 4.0e-9 ± 0.5e-10 2.1 ± 1.3

fH67R341A,H337A* 1.4e3, 1.7e3 4.6e-3, 6e-3 2.7e-6, 2.8e-6 2.3, 4.7

fH78 No binding detected

*values shown are from two independent experiments



Table 2 Data collection, phasing and refinement statistics (MIR)

Native P1

ESRF ID14eh4

Native C2

Diamond I03

Pt

Diamond I03

Hg

ESRF BM14

S-SAD

ESRF ID29

Data collection

Space group P1 C2 C2 C2 C2

Cell dimensions

a, b, c (Å) 48.7, 93.9, 99.7

185.4, 52.2, 128.7

186.8, 53.8, 129.5

186.1, 53.2, 128.4

187.7, 53.5, 130.2

α, β, γ (°) 80.1, 99.1, 84.8

90, 118.8, 90 90, 118.1, 90 90, 117.9, 90 90, 118.1, 90

Resolution (Å) 95-2.35(2.41-2.35)*

40-2.35(2.41-2.35)

115-3.2(3.3-3.2)

38-3.2(3.3-3.2)

40-3.0(3.1-3.0)

Rmerge 0.039(0.277) 0.046(0.316) 0.142(0.537) 0.070(0.095) 0.106(0.544)

I/σI 14.2(3.1) 20.6(4.5) 16.6(5.0) 14.9(9.9) 34.8(8.3)

Completeness (%)

91.3(80.9) 94.3(95.5) 99.3(99.0) 96.7(95.0) 99.8(99.0)

Redundancy 2.0(2.0) 3.6(3.7) 14(11.2) 3.8(3.8) 28.3(27.9)

Refinement

Resolution (Å) 95-2.35 40-2.35

No. reflections 64754 43159

Rwork/ Rfree 23.2/26.3 25.5/27.1

No. atoms

Protein 11434 8412

Water 508 424

B-factors

Protein 40 42

Water 44 39

R.m.s deviations

Bond lengths 0.007 0.012



(Å)

Bond angles (°)

0.99 0.94

*Highest resolution shell is shown in parenthesis. Data for each dataset were collected from a

single crystal



Figure 1: Sensorgram obtained with longer association phase for the interaction

between fHbp on the chip surface and fH67 in the fluid phase. To test the robustness of

the values obtained from fitting the data obtained with a ~1 minute association time (as in Fig

1) the SPR was repeated with a longer association phase (5 minutes). fH67 at concentrations

between 3 and 10nM (coloured lines, 3nM giving lowest signal, 10nM highest) was injected at

40µl/min and three independent series of eight injections were fit (black lines) to yield

estimates for the KD of 2.0±0.1nM (χ2 of 1±0.02) in good agreement with the statistics

reported from the shorter association time data presented in Fig 1 and Table S1. The residual

plot for the fit is shown in the lower panel and demonstrates that all residuals are <5.



Figure 2: Comparison of the structure of the BC region of fHbp to the earlier solution

structure. Overlay of NMR structure1 (pink backbone representation) for the BC portion of

fHbp (PDB ID: 1YS5) onto our structure of the extracellular portion of fHbp (A region yellow, B

region green and C region cyan). A “putty” representation is used to demonstrate thermal

mobility factors of the X-ray structure. Note the large divergence in structure between the X-

ray structure of the ABC portion and that of the B region of the NMR structure which adopts

an extended and partially helical structure which does not lie in any particular relationship to

the C-terminal β-barrel. These differences presumably reflect the fact that the BC construct

cannot fold correctly due to it containing only a portion of the N-terminal barrel.



Figure 3: Charge distribution on the interacting surfaces of fHbp and fH67. Charge

distributions calculated using the APBS module within PyMol7. In the upper panel fHbp is

shown as surface representation coloured by charge and fH67 as a grey ribbon, whilst in the

lower the representations are reversed. In each figure the orientation is so as to look onto the

binding site for the partner.



Figure 4: Flexibility in the fH67/fHbp complex. Overlay of the seven, crystallographically

independent copies of the fH67/fHbp complex from the two crystal forms (four copies of the

complex in the P1 form and 3 copies in the C2 form). The high degree of similarity despite

very different packing environments indicates that the complex is biologically relevant.

Rearrangement of the proteins within the complex is limited to a small amount of movement

in the position of CCP7 of fH67 relative to CCP6 and fHbp. The loops of fHbp which pack

closest to CCP7 are also relatively mobile as indicated by their higher temperature factors

(displayed here as a “thicker” putty representation).

fH CCP6

fH CCP7

fHbp



Figure 5: Location of antibody epitopes mapped on the surface of fHbp. Residues in the

protective epitopes deCCPibed in 1,8,9 are shown in CPK representations (Glu 211, Arg 214

and Arg 269) coloured by element (C green, N blue, O red). Epitopes for antibodies which

block fH binding10 shown as CPK coloured according to colouring of fHbp (region “A” yellow,

“B” region green, “C” region cyan). Whilst none of these epitopes maps directly to the fH

recognition site they all lie sufficiently close that binding of the large antibody is likely to hinder

recognition of fH. The histidine sidechain at the polymorphic residue 402 of fH is also shown

in a CPK representation. Although this residue lies close to a secondary contact with fHbp

(around residues 103 to 108 – see Fig. 2 main text) the evidence from SPR of the two

polymorphic forms suggests that this is not a critical contact. This is also supported by the fact

that this region of the complex (both the GNA loops and the fH domain) is the most mobile

region seen with significant variation in the way the two proteins contact one another seen at

this point between the crystallographic independent copies of the complex (Fig. S3).

fH His 402

Jar3/Jar5 epitope GNA residues 186 & 188

Jar10/Jar11/Jar32/Jar35 epitopes GNA residues 239, 245, 257

Jar13 epitope GNA residue 281

“Protective” epitopes GNA residues 214-211 and 269



Figure 6: Comparison of the structures of fH67 in complex with fHbp and fH678 in

complex with SOS. Overlay of the seven, crystallographically independent copies of the

fH67 portion of the fh67:fHbp complex with the structure of fH6782 (in green, PDB ID:2UWN).

The position of CCP7 in the structure of fH678 is seen to lie in the middle of the range of

variation with respect to CCP6 seen in the structure of fH67 complexed to fHbp. The pattern

of thermal mobility factors is rather different (displayed as the thickness of the putty

representation) with the CCP6 helix that is buried in the fHbp complex seen to be less mobile

in the complex structure than in the earlier structure of fH678 in complex with sucrose

octasulphate.

fH CCP6 fH CCP7 fH CCP8



Figure 7: SOS inhibits binding of fH to N. meningitidis expressing fHbp on their

surface. Wild-type MC58 (fHbp+) or an isogenic mutant lacking fHbp (fHbp-) were incubated

with purified fH (225 mg/ml) with or without SOS as indicated. Binding was measured by

FACS analysis with an anti-human fH pAb and expressed as the mean fluorescence index

(MFI). SOS (100 mM, final concentration) inhibited binding of full length fH to bacteria

(Student’s T test, p<0.001).



Figure 8: Heparin inhibits binding of fH to fHbp. As for SOS (Fig 3d) heparin (SIGMA,

average molecular weight 18 kDa) inhibits the interaction of fH67 with fHbp with a

concentration of 1mM decreasing binding by ~50%.



Figure 9: Binding of full length fH to fHbp assayed using SPR. Using full length fH

purified from serum by an anti-fH column, binding to fHbp on the chip surface can be clearly

detected with fH at concentrations in the range 10-50 nM (coloured lines, 10nM lowest trace,

50nM highest, mock-coupled control subtracted). These data cannot be fit using simple

models to derive kinetic or affinity parameters probably due to factors including the fact that

the large size and inherent flexibility of the 20 CCP long fH will complicate the interaction

kinetics and in particular may impede diffusion of fH through the dextran surface of the sensor

chip to allow access to the coupled fHbp so that the apparent affinity is lower than the true

affinity.



Figure 10: Comparison of the structures of fHbp with that of an engineered avidin SSM4

was used to overlay secondary structure elements (cutoff of 40% match) between the

structure of fHbp (green) and the earlier structure of an engineered avidin5 (blue). Although

the topology of the N-terminal β-barrel of fHbp is very different to that of the avidin barrels, the

relative arrangement of the pairs of barrels is seen to be grossly similar. This suggests that

packing of two β-barrels selects for particular crossing angles for maximum stability.



Figure 11: Comparison of the structure of fHbp with that of an complement Factor B

SSM was used to overlay secondary structure elements between the structure of fHbp

(green) and the earlier structure of complement Factor B 6 (red for overlaid portion and grey).

Although the topologies of the β-barrels are different, the relative arrangement of the pairs of

barrels is seen (again) to be grossly similar.



Supplemental References

1. Cantini, F. et al. Solution structure of the immunodominant domain of

protective antigen GNA1870 of Neisseria meningitidis. J Biol Chem 281,

7220-7 (2006).

2. Prosser, B.E. et al. Structural basis for complement factor H linked age-related

macular degeneration. J Exp Med 204, 2277-83 (2007).

3. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison.

Trends Biochem Sci 20, 478-80 (1995).

4. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool

for fast protein structure alignment in three dimensions. Acta Crystallogr D

Biol Crystallogr 60, 2256-68 (2004).

5. Hytonen, V.P. et al. Controlling quaternary structure assembly: subunit

interface engineering and crystal structure of dual chain avidin. J Mol Biol

359, 1352-63 (2006).

6. Milder, F.J. et al. Factor B structure provides insights into activation of the

central protease of the complement system. Nat Struct Mol Biol 14, 224-8

(2007).

7. DeLano, W.L. on World Wide Web http://www.pymol.org(2002).

8. Giuliani, M.M. et al. The region comprising amino acids 100 to 255 of

Neisseria meningitidis lipoprotein GNA 1870 elicits bactericidal antibodies.

Infect Immun 73, 1151-60 (2005).

9. Welsch, J.A., Rossi, R., Comanducci, M. & Granoff, D.M. Protective activity

of monoclonal antibodies to genome-derived neisserial antigen 1870, a

Neisseria meningitidis candidate vaccine. J Immunol 172, 5606-15 (2004).

10. Beernink, P.T. & Granoff, D.M. Bactericidal antibody responses induced by

meningococcal recombinant chimeric factor H-binding protein vaccines. Infect

Immun 76, 2568-75 (2008).



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Neisseria meningitidis recruits factor H using protein ... · Supplemental Methods for Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates Muriel