Polar contacts in the model between 17.2 and β1-AR.

<p>MC: Main Chain; SC: Side Chain.</p

Structure of the Fab17.2 – R13 complex.

<p>A. Superposition of the apo Fab 17.2 and R13 complex structures (grey and green respectively). VH and VL contact residues are indicated. B. Main water molecules present on the antigen binding site of Fab 17.2 apo. C. Superposition of water molecules present in the apo Fab 17.2 that are replaced by the peptide in the Fab 17.2 R13 complex. D. Superposition of R13 peptides from molecules 1 (green) and 2 (magenta). E. Structure of the Fab 17.2-R13 complex (molecule 1). All hydrogen bonds between mAb 17.2 and peptide R13 are illustrated as dotted yellow lines. The π-stacking interaction between VL Tyr101 and the epitope Phe9 is also indicated. Heavy chain CDRs are coloured in red and light chain CDRs in blue the peptide is coloured in light green.</p

Buried surface upon complex formation, with shape complementarities (Sc) [50] and number of contacts observed between the mAb 17.2 and the R13 peptide in the crystal structure¹ and the model².

<p>Buried surface upon complex formation, with shape complementarities (Sc) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001375#pntd.0001375-Lawrence1" target="_blank">[50]</a> and number of contacts observed between the mAb 17.2 and the R13 peptide in the crystal structure¹ and the model².</p

Polar contacts between Fab 17.2 and R13 peptide.

<p>MC: Main Chain; SC: Side Chain.</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

Model of the interaction of Fab 17.2 with the human β1-AR.

<p>A. Superposition of the second extracellular loop (2ECL) of the human β1-AR model (violet) and the region 2–5 of the epitope (light green). B. Complex of the Fab 17.2 (light green) with the human β1-AR (violet) inserted in a membrane model (grey). C. Zoom of the paratope region of 17.2 interacting with the second extracellular loop. The main contact points are illustrated as dotted yellow lines. Heavy chain CDRs are coloured in red and light chain CDRs in blue.</p

Functional activity of the mAb 17.2.

<p>A. Representative histogram showing the HEK and HEK-β1 cells labelled with mAb 17.2 followed by a Cy3-conjugated goat anti-mouse IgG. B. Results are expressed as means ± SD (n = 3). Inset: Binding of mAb 17.2 to HEK-β1 cells in the presence of R13 peptide. ** <i>p</i><0.01; *** <i>p</i><0.001. C. Passive transfer of mAb 17.2 to naïve mice. Repolarisation abnormalities (a) and first degree AV conduction block (b) are indicated by arrows. bpm: beats per minute.</p

Structure of the Fab 17.2.

<p>A. Apo Fab 17.2 structure (molecule 1). Heavy chain CDRs are coloured in red and light chain CDRs in blue. B. Superposition of molecules 1, VH-VL region of the two crystals asymmetric units. C. Superposition of molecules 2, VH-VL region of the two crystals asymmetric units. Apo Fab 17.2 (grey), Fab 17.2-R13 molecule 1 (green) and molecule 2 (magenta).</p

scFv C5 intrabody expression in T. <i>brucei</i>.

<p><b>A.</b> Western blot detection of C5 intrabody expression at 0, 24 and 72 hours in six different clones. C5 was detected by an anti-Myc antibody. The loading control ALD was detected by an anti-aldolase antibody. <b>B.</b> Averaged densitometry analysis of intrabody expression (***; p<0.001). <b>C.</b> Averaged growth rate curve of the six different clones induced and un-induced C5 intrabody parasites (***; p<0.001).</p

Translation inhibition.

<p><b>A.</b> Crude extracts from <i>T. cruzi</i> (lane 1), <i>T. brucei</i> (lane 2), <i>C. fasciculata</i> (lane 3) and <i>R. norvegicus</i> (lane 4) were analyzed by SDS-PAGE and Western blot with scFv C5. The position of previously characterized P proteins from <i>T. cruzi</i> (Tc) is shown on the left. The image is representative of three independent assays. <b>B.</b> Dose-response effect of scFv C5 on protein synthesis by ribosome extracts from <i>R. norvegicus</i> and <i>T. cruzi</i>. <b>C.</b> Effect of scFv C5 160 nM on <i>in vitro</i> protein synthesis in ribosome extracts from <i>R. norvegicus</i>, <i>C. fasciculata</i> and <i>T. cruzi</i> compared with the translation inhibitor emetine at 0.1 mg/mg. Average values for control assays were 80,000 cpm, 48,000 cpm and 43,000 cpm for <i>R. norvegicus</i>, <i>C. fasciculata</i> and <i>T. cruzi</i>, respectively. The scFv C5 significantly inhibited protein synthesis by <i>C. fasciculata</i> and <i>T. cruzi</i> ribosomes (*, p<0.05; ***, p<0.001). <b>D.</b> Effect of the preincubation with R13 or H13 peptide on the translation inhibition by scFv C5 (**; p<0.01).</p