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Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin - PubMed

  • ️Wed Jan 01 2020

Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin

Kartik Manne et al. Acta Crystallogr D Struct Biol. 2020.

Abstract

BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.

Keywords: BibA; C4b-binding proteins; group B streptococcus; immunogenic bacterial adhesins; three-helix-bundle-motif repeats.

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Figures

Figure 1
Figure 1

(a) Predicted domain arrangement for the GBS surface protein BibA. The C-terminal sorting motif is shown. Different C4BP variants were used: plasma-purified C4BP (b), rC4BP (c) and C4BP α-chain deletion mutants (d). The main isoform of C4BP contains seven identical α-chains and one unique β-chain. The β-chain-containing C4BP in circulation is bound to the vitamin-K-dependent anticoagulant Protein S (PS), forming a C4BP–PS complex. The rC4BP contains six α-chains and lacks the β-­chain and the associated PS. C4BP α-chain deletion mutants lack single CCP domains (represented by gray circles with an X in each α-chain).

Figure 2
Figure 2

(a) Circular dichroism spectra of freshly prepared and intact BibA34–400 sample at 20°C (red), at 40°C (orange) and denatured at 80°C followed by cooling to 20°C (green), the standard deviation error between the BibA34–400 samples (gray dotted line) and the spectrum generated from the BibA126–398 crystal structure (black). Two negative peaks at 208 and 222 nm typical of a α-helical secondary structure were observed for the BibA34–400 sample. (b) Estimated secondary-structure content (%) of BibA34–400 sample in solution (top) and the spectrum generated from the BibA126–398 crystal structure (bottom).

Figure 3
Figure 3

(a) A schematic of GBS BibA34–400 with N-terminal His tag and TEV protease cleavage site. (b) SDS–PAGE gel showing the breakdown of the BibA34–400 sample. The marker, BibA34–400 without any mutations and BibA34–400 with K49N, K70N and K112N mutations are labeled M, 1 and 2, respectively. (c) Picture of a BibA126–398 crystal formed after proteolysis.

Figure 4
Figure 4

(a) Cartoon representation of the BibA126–398 crystal structure shown as a rainbow model with color changing gradually from the N-­terminus (blue) to the C-terminus (red). The length of the rod-shaped structure is depicted, and the antiparallel three-helix-bundle-motif repeats are labeled from the N-­terminus to the C-terminus as MR1–MR4, respectively. (b) Superposition of the antiparallel three-helix-bundle-motif repeats MR1 (dark blue), MR2 (cyan) and MR3 (forest green). Similar residues such as Leu and Ile are boxed and shown in a red font and the conserved serine residue is highlighted in red.

Figure 5
Figure 5

(a) Pair distance distribution [P(r)] function of intact BibA34–400 protein. (b) Comparison of the experimental scattering profile (in blue) for BibA34–400 with profiles from a theoretical model (FoXS; green) derived from the proposed BibA34–400 model. (c) Fit of the crystal structure of BibA126–398 (cyan) and the proposed BibA34–400 (magenta) into the ab initio model of BibA34–400 calculated with DAMMIF.

Figure 6
Figure 6

Microtiter plates were coated with BibA (5 µg ml−1) and increasing amounts of plasma-purified C4BP (a) or rC4BP (b) were added. Binding was detected using specific polyclonal anti-C4BP antibodies. BSA was used as a negative control. The mean ± SD of three independent experiments performed in duplicate is presented. Statistical significance was calculated using a two-way Anova test. ***, p < 0.001. (c) Surface plasmon resonance analysis to measure the binding of BibA to immobilized C4BP using Biacore. (d) The C4BP variants were allowed to bind to BibA immobilized on a plate. Bound C4BP was detected with polyclonal Abs. The graph represents data from three independent experiments performed in duplicate ± SD. Statistical significance was calculated using a one-way Anova test. *, p < 0.05; ***, p <0.001.

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