Structure of factor H-binding protein B (FhbB) of the periopathogen, Treponema denticola: insights into progression of periodontal disease

Abstract

Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A K(D) value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.

FIGURE 1.

Structure and physiochemical properties of the T. denticola FH-binding protein, FhbB. A, FhbB (ribbon diagram) is color-coded as follows: α-helix 1, green; turn 1, yellow; α-helix 2, light blue; β-strand 1 (orange); turn 2, black; α-helix 3, dark blue; and β-strand 2, red. The N and C termini of the protein are indicated. The right image is rotated 180° around the y axis. B, electrostatic surface map of FhbB. The left and right images are oriented as in A. Positive and negative charges are indicated by blue and red, respectively. C, hydrogen bonding salt bridge network between turns 1 and 2 is depicted by dashed red lines with the distance indicated in angstroms. Residue numbering is based on the full-length FhbB sequence of T. denticola strain 35405. D, hydrophobic core of FhbB. The polypeptide backbone is indicated in gray, and the orientation of the protein is as in B. The atomic composition of the core is indicated as follows: carbon (teal), sulfur (orange), oxygen (red), and nitrogen (blue).

FIGURE 2.

FhbB is a highly thermostable protein. The thermostability of FhbB was assessed by circular dichroism. CD spectrum of FhbB (A) and lysozyme (C) was measured at 4 °C prior to heating and compared with the spectrum after heating the protein to 90 °C and returning to 4 °C (black, spectra prior to heating the protein; gray, spectra after heating and cooling the protein). α-Helical content of FhbB (B) and lysozyme (D) was monitored throughout the heating and cooling by measuring the CD at 222 nm (black, heating from 4 to 90 °C; gray, cooling from 90 to 4 °C).

FIGURE 3.

Analysis of FhbB dimerization. A, center figure of A depicts the dimeric FhbB asymmetric unit with the water molecules at the interface indicated by blue spheres. To the left and right, the component monomers are shown, indicated in light green and yellow, after rotation 90° to allow for visualization of the buried interface and hydration patterns. B, interface is enlarged with interface residues indicated. C provides a top down view of the interactions at the interface. Residues involved in hydrophobic interactions are indicated with black dashed lines (distances in Å). D, sedimentation coefficients of recombinant FhbB over a concentration range of 13–821 μm were determined (monomer, 1.31 S; dimer, 2.2 S).

FIGURE 4.

Site-directed mutagenesis of FhbB and quantification of FH binding. A, ribbon diagrams with superimposed transparent electrostatic surface charge maps are presented. The spatial placement of residues targeted for substitution are indicated on the appropriate face of the FhbB monomer. B, measurement of FH binding to wild type and single and double amino acid FhbB substitution mutants using ELISA. Recombinant proteins were immobilized in the wells, and purified human FH was added and binding assessed using HRP-conjugated anti-FH antibody. Each mutant is indicated by the x-axis, and the y axis indicates the percentage of binding relative to WT. Values represent the mean calculated from three independently performed experiments ± S.E. (*, p < 0.01; **, p < 0.001). C, analysis of FH binding to select FhbB substitution mutants using affinity ligand binding immunoblot assay approach. Purified recombinant proteins (indicated above each lane) were fractionated by SDS-PAGE and transferred to membranes. The membranes were incubated with FH, and then FH binding was assessed using HRP-conjugated anti-FH antibody. Equivalent loading of recombinant protein in each lane was verified by immunoblotting using anti-FhbB antiserum. D, determination of affinity constants through surface plasmon resonance. No binding of FH to the E45A, D58A, and E62A substitution mutants was detected, and hence affinity constants were not determined.

FIGURE 5.

Interaction of FhbB with FH. A, membrane-bound recombinant FhbB, B. hermsii (Bh) FhbA (positive control for binding to CCPs19–20), and B. burgdorferi (Bb) BBK32 (negative control) were tested for their ability to bind full-length FH or fragments consisting of CCP6–8 and CCP19–20. Binding was assessed using an overlay assay. B, ability of SOS to inhibit FH binding to FhbB was assessed. ELISA demonstrates that FH binding to FhbB is inhibited by increasing concentrations of SOS.