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

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

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

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

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

Structural Basis for Group B <em>Streptococcus</em> Pilus 1 Sortases C Regulation and Specificity

<div><p>Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B <em>Streptococcus</em> (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. <em>In vitro</em> experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, <em>in vitro</em> FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.</p> </div

Sequences of fluorescent peptides used in this study.

<p>Sequences of fluorescent peptides used in this study.</p

Structural differences of NadR in ligand-bound or free forms.

<p><b>(A)</b> Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. <b>(B)</b> Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. The inner conformer is the one that would display major clashes if 4-HPA were present. <b>(C)</b> Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation.</p

Structural comparisons of NadR and modelling of interactions with DNA.

<p><b>(A)</b> The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. <b>(B)</b> The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. <b>(C)</b> The double-stranded DNA molecule (grey cartoon) from the OhrR-<i>ohrA</i> complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. The proteins share ~30% amino acid sequence identity. For clarity, only the α4 helices are shown in panels (B) and (C). <b>(D)</b> Upon comparison with the experimentally-determined OhrR:<i>ohrA</i> structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove.</p