Table 1.
Data collection and refinement statistics.
Fig 1.
Overall structure and topology of the backbone pilus protein BP-2b.
(A) The structure of BP-2b is depicted with light blue and orange cartoons for domains D2 and D3. Intradomain isopeptide bonds in D2 and D3 are shown with sticks, while green spheres show calcium ions located on the surface. The red dashed lines indicate the only residues that could not be modelled due to the lack of electron density. (B) Cartoon representation of D2 and D3 of BP-2b, with the two domains reoriented with respect to the view in A), and colored with a gradient according to the topology diagram on the right. (C) Topology diagram for D2 and D3 of BP-2b, with their core β-strands labeled A—G in rainbow style colored as in panel B. The strands linked by isopeptide bonds in D2 and D3 are marked by thick horizontal black lines.
Fig 2.
Domains D2 and D3 are colored as in Fig 1 in blue and orange, respectively. Isopeptide bonds between Asn330 and Lys 187 for D2, and between Asn462 and Lys358 in D3 are shown with blue and orange sticks, and 1σ 2Fo-Fc electron density map around this region is shown as blue mesh (carve = 1.1). The magenta colored regions in (A) and (B) show the location of hydrophobic residues surrounding the isopeptide bonds. Hydrogen-bonds between the isopeptide bonds and the nearby Asp (225, D2) and Glu (423, D3) are shown with black dashed lines.
Table 2.
Multiple structural alignment of BP-2b protein with other known structures using the DALI server.
Fig 3.
Structural comparisons of BP-2bD2+D3 with other pilin backbone proteins.
(A) BP-2b (blue cartoon) is shown overlaid onto: the pilus backbone protein RrgB (pdb 2x9x, red cartoon, left), the major pilin protein GBS80 (pdb 3pf2, green cartoon, middle), and on the major pilin protein BP-2a (pdb 2xtl, pink cartoon, right). (B) Domain architecture of GBS backbone proteins from pilus 1 (BP-1), pilus 2a (BP-2a) and pilus 2b (BP-2b). The proteins are comprised of a signal peptide (SP) at the N-terminus and a C-terminal LPXTG-like motif (in red) linked to the transmembrane domain (TM). BP-1 and BP-2b contain three domains, while BP-2a four domains. The pilin motif involved in pilus polymerization is located near the D1–D2 domain linker while the E-box is located close to the sorting signal. Residues involved in isopeptide bonds are indicated by black bars. Domains present in the crystal structures are included into the box outlined with dashed lines.
Fig 4.
Lys 175, Glu 423 and the sorting motif LPSTG are involved in BP-2b polymerization in GBS.
Immunoblot analysis of total protein extracts from GBS mutant strain lacking the pilus 2b backbone protein gene (ΔBP-2b) complemented with plasmids expressing the wild-type BP-2b protein (WT) or BP-2b mutants carrying a deletion of the C-terminal sorting signal (BP-2bΔLPXTG), alanine substitutions of the putative pilin motif lysine (BP-2bK175A, BP-2bK118A BP-2bK82A) or of the E-box E423 residue (BP-2bE423A). Nitrocellulose membrane was probed with a mouse antiserum raised against the recombinant BP-2b protein (α-BP–2b).
Fig 5.
Biochemical characterization of different BP-2b constructs.
(A) Time course of the trypsin-proteolysis reactions at 37°C of BP-2b full length and fragments, analyzed by SDS-PAGE. Different digestion patterns can be observed for the different constructs. Asterisks indicate the not-digested proteins. (B) Differential Scanning Fluorimetry (DSF) analysis of BP-2b proteins (D1+D2+D3, D2+D3 and single domains D1, D2, D3) in presence of Sypro orange showed different thermal stabilities. Graph shows the fluorescence intensity vs. the temperature for the unfolding different BP-2b constructs. (C) Correlation of BP-2b melting temperature with the concentration of Ca2+.
Table 3.
Neonatal protection conferred by BP-2b domains vs the full length protein against GBS strain COH1 assessed by active maternal mouse immunization/neonatal pup challenge model.