Comparison of β-strand organization in three CnaB-like pilin domains.

<p>Stereo figure shows a superposition of strands A, F and G from RrgB D1 (wheat), SpaA D1 (green) and BcpA D2 (grey) showing the divergence of strand G observed in RrgB D1. The positions of the YPKN pilin motif residues are indicated. RrgB D1 and SpaA D1 are the N-terminal domains of their respective proteins and both have a pilin motif (YPKH for SpaA D1); their superposition shows that two possible orientations are possible for strand G in such domains. SpaA D1 has no internal isopeptide bond is it lacks the essential Lys-Glu-Asn triad. In BcpA D2, which has no pilin motif, strands A and G stay together and a Lys-Asn isopeptide bond is formed (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022095#pone-0022095-g002" target="_blank">Fig. 2</a>).</p

β-sheet topology and isopeptide bond formation.

<p>A) Topological representation of domain D1 of RrgB. The final β-strand G carries the YPKN pilin motif residues, which include the lysine used for ligation to the next subunit. In RrgB D1, strand G diverges from the N-terminal strand A at the proline residue of this motif, transferring its hydrogen bonding to strand F. B) In a conventional CnaB domain, which comprises a 3-stranded β-sheet D-A-G and a 4-stranded sheet F-E-B-C and strand G is hydrogen bonded to strand A along its whole length, allowing an isopeptide bond to be formed between a lysine in strand A and an asparagine in strand G. C) Electron density for the YPKN motif (residues 181–184) in β-strand G of domain D1 together with that for the other two residues that could potentially participate in internal isopeptide bond formation, Lys41 and Glu143. The electron density is from a 2Fo – Fc map contoured at 0.38 eÅ⁻³ (1.5σ). D) Superposition of strands A and E of RrgB D1 (wheat) on to the equivalent strands of domain D2 of BcpA from <i>B. cereus</i> (green) showing that an isopeptide bond such as that present in BcpA between Lys174 and Asn265 cannot occur in RrgB D1 because the divergence of strand G in RrgB D1 pulls Asn184∼4 Å further away from the lysine (Lys41) in strand A.</p

Model for RrgB polymer formation in pneumococcal pili.

<p>Cartoon and surface representation of showing the docking of two successive molecules of full-length RrgB. Model is based on the molecular packing observed in crystals of RrgB, but with successive molecules rotated ∼180° in order to conform to the cryo-EM density observed for pneumococcal pili <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022095#pone.0022095-Hilleringmann1" target="_blank">[27]</a>. In this model, the C-terminal residue Thr627 of the crystal structure is ∼19 Å from the ε-amino group of Lys183, a distance that would accommodate the four additional residues 628–631 to the true C-terminus. The enlarged area shows a surface groove into which the missing residues could fit, running from the C-terminal Thr 627 (blue) to Lys183 from domain D1 (red).</p

Structure of full-length RrgB.

<p>Cartoon representation of the three-dimensional structure of the major pneumococcal pilin RrgB, with the four domains labelled from D1 (N-terminal domain) to D4 (C-terminal domain). β-strands are coloured in wheat and α-helices in blue. In D1, the section of β-strand G that contains the YPKN pilin motif is in green, with the side chains of Lys183 and Asn184 also in green, in stick mode. The side chain of Lys41 from β-strand A in D1 is in magenta, in stick mode. The isopeptide bond crosslinks in domains D2, D3 and D4 are shown in red, in stick mode, and the Lys and Asn residues involved in each case are indicated. The N- and C-termini of the polypeptide chain are also identified.</p

Structures used for docking to MenH.

<p>Chemical structures of the substrate (SEPHCHC) and transition state (TS) models used in docking studies to MenH. Bonds marked with a curly arrow were defined as rotatable in GOLD, and the atoms marked with an asterisk were able to undergo ring flipping.</p

Sequence alignment of Spy0129 with other class B sortases.

<p>Invariant residues are highlighted in dark blue and conserved residues in lighter blue colours. Putative catalytic residues are coloured in purple. The secondary structure elements from Spy0129 are shown above the sequence. Residues corresponding to the sortase signature motif are boxed with a black outline. The β6/β7 loop region of <i>S. aureus</i> SrtB which was shown to confer substrate specificity is outlined with a dotted red box.</p

SEPHCHC substrate of the MenH reaction.

<p>The succinyl and enol pyruvate groups are highlighted in boxes. Numbering refers to the carbons on the ring.</p

Comparison of different sortase structures.

<p>A) Class B: Spy0129 from <i>S. pyogenes</i> (this work); B) Class B: SrtB from <i>S. aureus</i> (PDB code 1T2P): C) Class C: SrtC1 from <i>S. pneumoniae</i> (PDB code 2W1J); and D) Class A: SrtA-LPAT* complex structure from <i>S.aureus</i> (PDB code 2KID). In each case, the β6/β7 region is highlighted with darker colour than the rest of the molecule. The lockable lid in the pneumococcal SrtC1 is highlighted in blue. All four structures are shown in equivalent orientations. The catalytic Cys residues are shown in stick mode.</p

Refinement Statistics.

A<p>Ramachandran regions and scoring as defined in Molprobity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061325#pone.0061325-Chen1" target="_blank">[26]</a>.</p>B<p>DPI Cruickshank’s DPI for coordinate error based on <i>R</i> factor (Å) from Buster 2.10.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061325#pone.0061325-Bricogne1" target="_blank">[24]</a>.</p

The EcMenH structure.

<p>(A) Topology diagram (B) Semi-transparent surface representation overlaying a cartoon representation as shown in c. Sulfate and chloride ions in the active site are depicted as sticks and a green sphere respectively (C) Cartoon representation. Helices and β-strands are labeled H and S respectively, secondary structure elements of the core α/β domain are colored red (helices) and yellow (β-strands) and those of the helical lid domain are in blue. The catalytic triad is depicted in stick representation in magenta.</p