Comparison of <i>Tco</i>ALD to other trypanosomatid aldolases.

<p>(A.) Sequence alignment between <i>Tco</i>ALD, <i>Tb</i>ALD, and <i>Lm</i>ALD. The colored bars above the sequence alignment represent the percentage of sequence identity: green (100%), green-brown (between 30% and 100%), and red (below 30%). The corresponding secondary structure elements are shown below the sequence alignment. Cylinders and arrows represent α-helices and β-sheets, respectively. The <i>Tco</i>ALD residues involved in the interaction with Nb474 are marked by an asterisk ‘*’. The residues highlighted in blue were selected for site-specific mutagenesis. (B.) Stereo view of a superposition of the crystal structures of the Nb474-<i>Tco</i>ALD complex (this work, color code as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.g002" target="_blank">Fig 2</a>), <i>Tb</i>ALD (PDB ID: 1F2J, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref026" target="_blank">26</a>], depicted in cyan), and <i>Lm</i>ALD (PDB ID: 1EPX, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref026" target="_blank">26</a>], depicted in blue). Those residues that are located in the vicinity of or on the <i>Tco</i>ALD epitope recognized by Nb474 and are distinct between the three trypanosomatid aldolases are shown in stick representation.</p

Homologous and heterologous sandwich ELISAs on the <i>Tco</i>ALD variants.

<p>(A.) Homologous sandwich ELISA on the <i>Tco</i>ALD variants in which His-tagged (Nb474H) and biotinylated (Nb474B) Nb474 were employed as capturing and detecting antibodies, respectively. (B.) Heterologous sandwich ELISA on the <i>Tco</i>ALD variants in which biotinylated Nb474 (Nb474B) and mouse anti-His IgG were employed as capturing and detecting antibodies, respectively.</p

A heat map readily identifies the optimal practical set-up to conduct the Nb474-based homologous immunoassay.

<p>Using a checkerboard system, varying amounts of capturing and detecting Nb474 were employed to identify those conditions yielding the highest ELISA signal. The details of this approach are described in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.ref016" target="_blank">16</a>].</p

Data collection and refinement statistics.

<p>Statistics for the highest resolution shell are shown in parentheses.</p

Investigation of the interaction between Nb474 and the <i>Tco</i>ALD variants by SPR.

<p>(A.-D.) SPR data recorded for the Nb474-<i>Tco</i>ALD^WT (A.), Nb474-<i>Tco</i>ALD^A77E (B.), Nb474-<i>Tco</i>ALD^L106Y (C.), and Nb474-<i>Tco</i>ALD^A77E/L106Y (D.) interactions with Nb474 as ligand and the <i>Tco</i>ALD variants as analytes. For <i>Tco</i>ALD^WT the sensorgrams are shown in orange, while the experimental traces are displayed in grey for the <i>Tco</i>ALD mutants. In all panels, the sensorgram in black indicates the concentration of analyte used to saturate the Nb474-coated sensor chip for the SPR sandwich assay. (E.-H.) Results of the SPR sandwich assay for the Nb474-<i>Tco</i>ALD^WT-Nb474 (E.), Nb474-<i>Tco</i>ALD^A77E-Nb474 (F.), Nb474-<i>Tco</i>ALD^L106Y-Nb474 (G.), and Nb474-<i>Tco</i>ALD^A77E/L106Y-Nb474 (H.) interactions with Nb474 as ligand and the <i>Tco</i>ALD variants as analytes. Upon saturation of the Nb474-coated sensor surface with analyte (saturation obtained after ~500 s and indicated by the black rectangle), varying concentrations of Nb474 are injected onto the sensor chip surface. In all panels, the resulting sensorgrams (black traces) are shown in the insets. For the Nb474-<i>Tco</i>ALD^WT-Nb474 (E.) and Nb474-<i>Tco</i>ALD^A77E-Nb474 (F.) sandwiches the additional binding of Nb474 was fitted with a 1:1 Langmuir binding model (red traces). The concentrations for all <i>Tco</i>ALD variants are expressed in terms of monomer concentrations.</p

Investigation of the stoichiometry of the Nb474-<i>Tco</i>ALD complex by analytical SEC.

<p>(A.-G.) Analytical SEC on the purified <i>Tco</i>ALD (A.), Nb474 (B.) and samples containing <i>Tco</i>ALD and Nb474 mixed at different molar ratios: 4:1 (C.), 4:2 (D.), 4:3 (E.), 4:4 (F.), and 4:6 (G.). All experiments were performed on a Superdex 200 HR 10/30 column. The black and grey traces represent the chromatograms of the different protein samples and the BioRAD gel filtration standard, respectively. In all figures, the inset shows an SDS-PAGE analysis of the elution peaks. <i>Tco</i>ALD (MM = 42.6 kDa) and Nb474 (MM = 15.8 kDa) are indicated by the orange and black arrows, respectively. <i>Lane M</i>, Prestained Protein Molecular Weight Marker (Fermentas). (H.) The calibration of the Superdex 200 HR 10/30 column that allows estimation of molecular mass based on the sample’s elution volume. The values between brackets indicate the estimated molecular mass versus the theoretical mass of the sample under investigation.</p

Kinetic parameters of the Nb474-<i>Tco</i>ALD^WT and Nb474-<i>Tco</i>ALD^A77E interactions.

<p>Kinetic parameters of the Nb474-<i>Tco</i>ALD^WT and Nb474-<i>Tco</i>ALD^A77E interactions.</p

Crystal structure of the (Nb474-<i>Tco</i>ALD)₄ complex.

<p>(A.) Overview of the entire complex. The <i>Tco</i>ALD tetramer and Nb474 are colored in orange and grey, respectively. The A and B dimer interfaces of <i>Tco</i>ALD are indicated, together with the distances between the Nb474 epitopes relative to these interfaces. (B.) Close-up of the interaction between Nb474 and a <i>Tco</i>ALD monomer. Color codes are as in (A.) and the Nb474 CDRs are indicated in different colors (CDR1, yellow; CDR2, green; CDR3, pink). (C.) Amino acid sequence of Nb474. The CDRs are highlighted in the same colors as in (B.). The residues marked by the asterisk ‘*’ are part of the Nb474 paratope and are involved in epitope binding. More details given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005932#pntd.0005932.s006" target="_blank">S1 Table</a>.</p