PfbAΔC SAXS data analysis.

<p>(A) GUINIER plot and residual of scattering data of PfbAΔC at 1.0 mg/ml are consistent with a radii of gyration of 34 Å. (B) The GNOM generated P(r) distribution of PfbAΔC at 1.0 mg/ml is asymmetric with a short tail extending towards a D_max approaching 120 Å. (C) Porod-Debye plots of PfbAΔC SAXS data at 1.0 mg/ml displays the characteristic asymptote of a well structured protein.</p

Schematic representation of the modular architecture of PfbA and structure of the parallel β-helix core domain.

<p>(A) The PfbA architecture include the FSIRK and LPXTG sequence motifs that are common Gram-positive export signal peptide and streptococcal cell-wall adherence features, respectively. The surface exposed α/β and cell-wall link domains are conserved across pneumococcal isolates. (B) Cartoon representation of the CAZyme-like 12-stranded β-helix domain solved by X-ray crystallography to 2.28 Å. The 90 residue (T139-Q230, coloured red) N-terminal initiator of the core domain is a conserved precursor of the parallel β-helix fold and includes the calcium-binding site established by the sidechains of E187 and H214 and the carbonyl oxygen of T225. A water coordinated by residues Q296, Q331, E333 and the carbonyl oxygen of G339 is reminiscent of the metal binding sites seen in polysaccharide lyases.</p

PfbAβ surface features and putative carbohydrate accommodation Cleft.

<p>(A) Carbohydrate accommodation groove of <i>endo</i>-<i>N</i>-acetylglucosaminidase tailspike protein from the <i>E. coli</i> bacteriophage HK620 complexed with the substrate <i>O</i>-antigen hexasaccharide (orange). (B) A putative carbohydrate accommodation groove (grey) formed along the length of the PfbAβ domain has features conserved across carbohydrate processing factors. (C) A series of electropositive residues (blue) line this cleft (dashed line) which could interact with negatively charged polysaccharides. (D) The solvent accessible surface of PfbAβ colored by electrostatic potential also reveals the electropositive nature of the cleft and shows the branched nature of the cleft. (E) Phylogenetic mapping of homologous sequences to PfbA. Conserved residues are shown in purple and pink, neutral in white, ambiguous in yellow, and non-conserved in blue as per Consurf standard colouration scheme. (F) The electronegative residues Q296, Q331, E333 and carbonyl G339 coordinate a crystallographic water, are conserved at the base of the cleft branch point. This putative active centre is lined with the electropositive series of residues H293, K320, K327, K338, H377 and R405 which have features similar to carbohydate active lyases. This conserved region is circled in panel E.</p

X-ray data collection, processing and PfbAβ model refinement statistics.

<p>X-ray data collection, processing and PfbAβ model refinement statistics.</p

SAXS statistics.

*<p>Theoretical molecular weight.</p>a<p>calculated by the method of Fischer <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067190#pone.0067190-Fischer1" target="_blank">[24]</a>.</p>b<p>average of χ-values determined for the 20 models calculated by the DAMMIF <i>ab initio</i> modelling procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067190#pone.0067190-Franke1" target="_blank">[25]</a>. Standard deviations calculated for the χ-values were <0.001.</p>c<p>averaged normalized spatial discrepancies (NSD) for the 20 models calculated by the DAMMIF <i>ab initio</i> modeling procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067190#pone.0067190-Volkov1" target="_blank">[44]</a>.</p

Overall model of PfbA.

<p>Utilizing a dissect-and-build approach of X-ray crystallography of the core domain (red, yellow and green) coupled with SAXs of the N-terminal (blue) and core domains, the overall structural features of the cell-wall attached pneumococcal adhesin PfbA reveal and overall elongated, bottle shaped structure. The cell-wall linking domain is schematically shown as tubes generally representing the predicted α-helical nature of this region. The putative carbohydrate recognition cleft is highlighted in grey.</p

PfbAΔC pseudo-atomic solution model.

<p>(A) Experimental and theoretical SAXS data calculated for the (B) averaged <i>ab initio</i> surface representation of 10 independent DAMMIF calculations and (C), rigid body fit of the PfbAβ domain into the envelope. (D) I-TASSER generated model of the PfbA N-terminal α/β domain (residues 54–138). (E) CRYSOL calculated and experimental SAXS data for the (F) PfbAΔC composite structure generated from SAXS data with the program BUNCH. Individual models resulted in excellent fits to the data with χ_(DAMMIF) values in the range of 1.5–1.6.</p

Microalgae produce DMSP; microbial lyases cleave DMSP into DMS, a process with relevant biogeochemical ramifications.

<p>(Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103128#pone.0103128-Curson1" target="_blank">[1]</a>). A complete depiction of catabolic processing of DMSP by microbes is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103128#pone.0103128-Reisch1" target="_blank">[6]</a>.</p

Dimerization shapes the tunnel active sites of <i>Rd</i>DddP with a binuclear iron metal center that is conserved in DddP lyases.

<p>(<b>a</b>) The dimer with one monomer in a cartoon plot presentation (beige, blue, green from the N-to the C-terminus), the second monomer in purple and the iron ions colored in orange. (<b>b</b>) The entrance to the two active sites and their path is indicated by the red arrows. (<b>c</b>) The metal binding site of <i>Rd</i>DddP with the two iron ions in orange and the conserved metal coordinating residues in grey. The distances between residues and metal ions, in angstroms, are indicated in red. (<b>d</b>) Comparison of the <i>Rd</i>DddP metal binding site (grey, iron ions as orange spheres) with the metal binding site of the methionine aminopeptidase (1MAT), (yellow sticks, cobalt ions as purple spheres). (e) Conservation of metal binding site and the putative catalytic residues in diverse DddP lyases from marine bacteria and fungi. Rd: <i>Roseobacter denitrificans</i> OCh 114, Rn: <i>Roseovarius nubinhibens</i> ISM, Sp: <i>Silicibacter pomeroyi</i> DSS-3, Rl: <i>Roseobacter litoralis</i> Och 149, Fg: <i>Fusarium graminearum</i> cc19, Ao: <i>Aspergillus oryzae</i> RIB40. Fc: <i>Fusarium culmorum</i>. Abbreviations in red indicate functionally characterized DddP lyases based on Todd <i>et al</i>. 2009. (notably here only a subset of DddP lyases is shown and the residues for metal binding and proposed catalysis are invariant in all DddP lyases presented in Todd <i>et al</i>. 2009). The alignment was prepared with ClustalW.</p

<i>Rd</i>DddP contains metal ions.

<p>(<b>a</b>) Purified <i>Rd</i>DddP enzyme at a concentration of ∼10 µM. (<b>b</b>) <i>Rd</i>DddP after removal of metal ions with chelex resin. (<b>c</b>) Plot of the abundance of transition metals in <i>Rd</i>DddP identified by ICP-MS.</p