Functional activity of the mutated proteins.

<p>Mutant proteins were tested for their ability to bind O⁺ RBCs and disrupt FCR3S1.2 rosettes. A) Representative dot plot of protein binding to RBC at the highest concentration (100μg/ml) from flow cytometric analysis. B) Proteins were tested in serial dilution from 100μg/ml to 3.06μg/ml for binding to RBCs. Results are presented as mean fluorescence intensity (MFI) fold increase over a negative control. Three independent duplicate experiments were performed. C) FCR3S1.2 trophozoite rosettes were mechanically disrupted and allowed to reform in presence of different concentration of mutant proteins. Proteins were tested at different concentration ranging from 400μg/ml to 5μg/ml. Three independent experiments were performed in duplicate, results shown are average ± SEM.</p

Figure S1 from Structural and functional studies of Spr1654: an essential aminotransferase in teichoic acid biosynthesis in <i>Streptococcus pneumoniae</i>

Absorption spectrum of Spr1654 and Spr1654-PLP. (a) Analysis of recombinant Spr1654-his (MW 47.7 kDa) by SDS-gel electrophoresis. The protein was separated by SDS-12% PAGE, followed by staining with Coomassie blue. Lane 1, Spr1654-his; M, molecular mass marker. (b) The absorption spectra of Spr1654 with (red line) and without PLP (blue line) were collected between 300 and 500 nm. The appearance of a 420nm peak indicates PLP binding to Spr1654

Localization of selected mutations on the protein molecular model.

<p>A) Domain organization of the IT4var60 PfEMP1 protein with underlined the construct used in this study. B) A molecular model of the NTS-DBL1α of IT4var60 was built based on the crystal structure of the NTS-DBL1α domain of PAvarO strain (PDB: 2yk0) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118898#pone.0118898.ref021" target="_blank">21</a>]. Three 90 degrees orthogonal views of the molecule in the cartoon representation with mutated amino acids depicted in color. Subdomains 1, 2 and 3 are colored in black, light brown and light blue, respectively. C) Three 90 degrees orthogonal views of the molecule surface in the surface charge potential representation. Arrows indicate positively charged patches Blue: positive charge; white neutral charge; red: negative charge. D) Three 90 degrees orthogonal views of the molecule in the surface representation with mutated amino acids depicted in color. Mut A (Y73A, K263E): orange; Mut B (K118E, G384H): green; Mut C (K202A, K206A): yellow; Mut D (K97A, K171A): purple; Mut E (K325A, K327A): red; Mut F (K97A): black; Mut G (K263E): blue; Mut H (K31A, K34A): cyan.</p

RBC and receptor binding site of NTS-DBL1α IT4var60.

<p>A) Molecular model of NTS-DBL1α IT4var60 showing the residues involved in RBC binding (Y73, K97 and K263), rosette inhibition (Y73 and K263) and heparin binding (K97). B) Comparison with previously identified residues that are important for BgA binding to the PAvarO variant (K179, K95, K166, R69: purple) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118898#pone.0118898.ref021" target="_blank">21</a>].</p

Figure S2 from Structural and functional studies of Spr1654: an essential aminotransferase in teichoic acid biosynthesis in <i>Streptococcus pneumoniae</i>

Residue Tyr84 in Spr1654 allows enough space for accommodating the N-acetyl group of the sugar substrate. Residues Y84 in Spr1654, W89 in ArnB and F84 in PseC are colored in cyan, orange and violet, respectively. PseC binds to PMP-UDP-4-amino-4, 6-dideoxy-L-AltNAc (PMP-UDP-L-AltNAc) and ArnB binds to UDP- 4-amino-4-deoxy-L-Arabinose (UDP- Ara4N)

Binding capacity and affinity of recombinant NTS-DBL1α (It4var60) variants to RBCs and ligands.

<p>Binding capacity and affinity of recombinant NTS-DBL1α (It4var60) variants to RBCs and ligands.</p

Mutated proteins binding and affinity to Heparin-FITC.

<p>Affinity of the mutated recombinant proteins for the ligand heparin was tested by microscale thermophoresis. Heparin FITC was kept at constant concentration of 100 nM while proteins were tested at ranging concentration varying between 0.5 to 60000 nM. Measurements were performed at 50% LED power and MST 60. Results were plotted using GraphPad Prism and K_D calculated using NanoTemper analysis software (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118898#pone.0118898.t001" target="_blank">Table 1</a>).</p

Expression of recombinant proteins in <i>E</i>. <i>coli</i>.

<p>A) Size exclusion chromatogram showing monomeric nature of wt and mutated proteins. B) The purity and quality of the mutants was assessed by electrophoresis: 2 μg of wt and mutant proteins were run on 12% SDS-PAGE gel under reducing conditions and stained with Coomassie. C) Far CD spectra of the proteins studied herein, showing nearly identical secondary structures for all the mutants. For color coding and mutation see Table I.</p

Crystal structures of H-2K^b in complex with gp34 and NY-gp34.

<p>Overall schematic views of the H-2K^b/gp34 and H-2K^b/NY-gp34 MHC complexes are presented in the left part of panels A and B. The α1, α2 and α3 domains of the MHC heavy chain are colored in white. The β₂m subunit is colored in green. The peptides are in blue. The 2F_o-F_c electron density maps for the peptides gp34 and NY-gp34 when bound to H-2K^b presented in the right part of panels A and B, respectively, are contoured at 1.0 σ. The final models are displayed for comparison. The peptides, depicted with their N-termini to the left and their C-termini to the right, are displayed ‘from above’ as seen by the TCRs.</p

Nitrotyrosination of p4Y in H-2D^b/NY-gp33 directly affects recognition by H-2D^b/gp33-specific TCR.

<p>Nitrotyrosination of the main TCR-interacting peptide residue p4Y will affect the structural conformation of both TCR interacting residues on H-2D^b and of the TCR P14. The peptide binding cleft of H-2D^b and the TCR, both colored in white, are annotated. Hydrogen bond interactions appear as dotted lines. <b>A.</b> In H-2D^b/gp33, the side chain of p4Y protrudes out of the H-2D^bpeptide-binding cleft, positioning itself perfectly in the hot spot of the p14 TCR composed of the CDR3 loops from both Vα and Vβ. It forms three hydrogen bonds, two of them directly with Y36(Vα) and G102(Vβ) on the TCR P14. The last hydrogen bond is formed with the side chain of the H-2D^b histidine residue H155, linking this domain of the heavy chain to the TCR. <b>B.</b> The side chain of the nitrotyrosinated p4-NY can not be accommodated within the hot-spot of P14, resulting in sterical clashes with the side chain of the TCR residue Y36(Vα). Furthermore, the negatively charged side chain of the H-2D^b residue E163, important for TCR recognition, would also be repelled by the introduced negatively charged nitrotyrosination. <b>C.</b> Similarly, the other rotamer of the nitrotyrosinated p4-NY would result in sterical clashes with both G102(Vβ)the side chain of H155, abolishing all formed hydrogen bond interactions.</p