Lys 175, Glu 423 and the sorting motif LPSTG are involved in BP-2b polymerization in GBS.

<p>Immunoblot analysis of total protein extracts from GBS mutant strain lacking the pilus 2b backbone protein gene (Δ<i>BP-2b</i>) 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-2b_ΔLPXTG), alanine substitutions of the putative pilin motif lysine (BP-2b_K175A, BP-2b_K118A BP-2b_K82A) or of the E-box E423 residue (BP-2b_E423A). Nitrocellulose membrane was probed with a mouse antiserum raised against the recombinant BP-2b protein (α-BP–2b).</p

Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

<div><p>Group B <i>Streptococcus</i> (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.</p></div

Biochemical characterization of different BP-2b constructs.

<p><b>(A)</b> 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>B)</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 <i>vs</i>. the temperature for the unfolding different BP-2b constructs. (<b>C)</b> Correlation of BP-2b melting temperature with the concentration of Ca²⁺.</p

Structural comparisons of BP-2b_D2+D3 with other pilin backbone proteins.

<p>(<b>A)</b> 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>B)</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.</p

Multiple structural alignment of BP-2b protein with other known structures using the DALI server.

<p>Hits are ranked by Z-Score with best hits at the top of the table.</p><p><i>PDB</i>: Protein Data Bank</p><p><i>rmsd</i>: root-mean-square deviation of Cα atoms of superimposed proteins in Angstroms</p><p><i>lali</i>: number of structurally equivalent positions</p><p><i>nres</i>: number of structurally equivalent aligned residues</p><p><i>%ide</i>: percentage of amino acid identity in aligned positions</p><p>Multiple structural alignment of BP-2b protein with other known structures using the DALI server.</p

Isopeptide bonds of BP-2b.

<p>Domains D2 and D3 are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125875#pone.0125875.g001" target="_blank">Fig 1</a> 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 2<i>F</i>o-<i>F</i>c 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.</p

FRET assay with PI-2a peptides for substrate specificity analysis of SrtA_ΔN40.

<p>The reaction solutions contained 256 µM of fluorescent peptide, 3 µM of SrtA_ΔN40 and 75 mM of triglycine as peptidoglycan analogue. The reactions were performed at 37°C in the assay buffer containing 20 mM HEPES, pH 7.5. We monitored fluorescence emission every 5 minutes and we observed an increase in fluorescence intensity only in the presence of AP2-2a peptide (filled squares), as opposed to BP-2a peptide (open squares) and AP1-2a peptide (filled triangles). In the presence of an additional variation of AP2-2a motif (AP2-2a_mut, open circles) the reaction did not occur.</p

Pilus proteins are mostly released into the supernatant in the AP2-2a_ΔLPKTG mutant strain.

<p>Proteins were collected from FMC culture supernatants (B and D) or harvested cell pellets (A and C) of GBS strain 515 wild-type (wt), 515 knock-out strain for AP2-2a (ΔAP2) gene and ΔAP2 strains complemented with pAM-AP2_ΔLPKTG and with pAM-AP2 wild-type. Protein fractions were analysed by immunoblot stained with antibody specific for AP2-2a (A and B) and for the backbone protein BP-2a (C and D). Asterisks (*) indicate the monomeric form of AP2-2a and BP-2a. The equal quantity loaded in each well is verified by immunoblotting the same gel with a control antiserum that recognizes the protein PcsB of 47 kDa (indicated by a black arrow).</p

Molecular mAbs/BP-2a 515 variant docking reveals two different binding orientations.

<p>(A) Ribbon representation of mAb 17C4/A3-BP-2a 515 variant complex. (B) Ribbon representation of mAb 4H11/B7-BP-2a 515 variant complex. In both complexes, the binding interface is CPK represented and colored according to the mAbs and antigen overall structure.</p

Protein sequences of the three functionally active mAbs.

<p>Sequence comparison between the functionally active mAbs selected against BP-2a protein 515 variant. CDR1, CDR2, and CDR3 as defined by Kabat <i>et al. </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003115#pcbi.1003115-Kabat1" target="_blank">[39]</a> are highlighted.</p