Polymorphisms found in the ARC domains contribute to L6 recognition strength.

<p><b>A and B</b> left panel: Schematic diagram of chimeric L5–L6 proteins. Domains from L6 are shaded white and domains from L5 are shaded grey. LRR units with polymorphic residues in the β-strand/β-turn structure (xxLxLxx motif) are marked with black bars. <b>A and B</b> right panel: Growth of yeast strain HF7c expressing chimeric L5–L6 proteins fused to the GAL4 activation domain and AvrL567-A and -D proteins fused to the GAL4 DNA-binding domain. Cultures were grown on selective media lacking tryptophan, leucine and histidine and scored after 4 days.</p

Homology model of the L6 NB-ARC domain.

<p>A homology model of the L6 NB-ARC domain was prepared using Modeller <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003004#ppat.1003004-Eswar1" target="_blank">[51]</a>. The NB sub-domain is coloured in yellow, the ARC1 sub-domain is coloured in green, and the ARC2 sub-domain is coloured in orange. <b>A.</b> Cartoon representation of the NB-ARC domain with the polymorphic residues A454, E457, E461 and R465 represented as sticks. <b>B.</b> Surface representation of the NB-ARC domain with the polymorphic residues in <b>A</b> highlighted in blue. The molecule is oriented as in <b>B.</b> The figure was prepared using PYMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p

Polymorphisms found in the LRR domain determine L5 and L6 recognition specificity.

<p><b>A and C.</b> Schematic diagrams of L6₅₉₂L5, L6₄₉₃L5 and L5₅₅₆L6 chimeric proteins. Domains from L6 are shaded white and domains from L5 are shaded grey. LRR units with polymorphic residues in the β-strand/β-turn structure (xxLxLxx motif) are marked with black bars. <b>B and D.</b> Growth of yeast strain HF7c expressing L6₅₉₂L5, L6₄₉₃L5 or L5₅₅₆L6 proteins fused to the GAL4 activation domain and wild-type (wt) and mutant AvrL567-A and -D proteins fused to the GAL4 DNA-binding domain. Cultures were grown on selective media lacking tryptophan, leucine and histidine and scored after 4 days.</p

L6₄₉₃L5₁₁₉₃L6 recognizes AvrL567-A and -D <i>in planta</i>.

<p><b>A.</b> Transgenic tobacco W38 expressing AvrL567-A, 4 days after infiltration with <i>A. tumefaciens</i> strains carrying L5, L6 or L6₄₉₃L5₁₁₉₃L6. <b>B.</b> W38 tobacco 4 days after co-infiltration with <i>A. tumefaciens</i> strains carrying L5, L6 or L6₄₉₃L5₁₁₉₃L6 and AvrL567-D. <b>C.</b> W38 tobacco 4 days after co-infiltration with <i>A. tumefaciens</i> strains carrying L5, L6 or L6₄₉₃L5₁₁₉₃L6 and AvrL567-A-R96S. <b>D.</b> W38 tobacco 4 days after co-infiltration with <i>A. tumefaciens</i> strains carrying L5, L6 or L6₄₉₃L5₁₁₉₃L6 and an empty vector.</p

Polymorphisms found in the N- and C-terminal LRR units are critical for AvrL567 specificity.

<p><b>A–E</b> left panel: Schematic diagram of chimeric L5–L6 proteins. Domains from L6 are shaded white and domains from L5 are shaded grey. LRR units with polymorphic residues in the β-strand/β-turn structure (xxLxLxx motif) are marked with black bars. <b>A–E</b> right panel: Growth of yeast strain HF7c expressing chimeric L5–L6 proteins fused to the GAL4 activation domain and AvrL567-A, -D, and -J proteins fused to the GAL4 DNA-binding domain. Cultures were grown on selective media lacking tryptophan, leucine and histidine and scored after 4 days.</p

The amino acid residues present at positions 50, 56, 90 and 96 in AvrL567 variant proteins is shown along with the L5, L6 and L6RVL11 recognition specificity observed in the yeast-two-hybrid assay.

<p>Italicized text indicates data from Wang <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003004#ppat.1003004-Wang1" target="_blank">[45]</a> or Dodds <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003004#ppat.1003004-Dodds2" target="_blank">[16]</a>.</p><p>−indicates no interaction, +indicates an interaction, +/−indicates a weak interaction.</p

Polymorphisms in L5 co-vary between the TIR domain and the N-terminal LRRs.

<p><b>A–C left panel:</b> Schematic diagram of chimeric L5–L6 proteins. Domains from L6 are shaded white and domains from L5 are shaded grey. LRR units with polymorphic residues in the β-strand/β-turn structure (xxLxLxx motif) are marked with black bars. <b>A–C right panel:</b> Growth of yeast strain HF7c expressing chimeric L5–L6 proteins fused to the GAL4 activation domain and AvrL567-A and -D proteins fused to the GAL4 DNA-binding domain. Cultures were grown on selective media lacking tryptophan, leucine and histidine and scored after 4 days.</p

Scatter plot of ω values of codons found in the TIR (grey box), NB (purple box), ARC1 (blue box), ARC2 (green box), spacer (SPCR; orange box) and LRR (red box) regions of <i>L</i> locus R proteins.

<p>Codons with statistically significant (<i>p</i>>0.95) ω values are represented by red dots with error bars representing the standard error of the mean. The percentage of statistically significant positively selected codons is indicated above each region. The dashed line indicates a ω value of 1.</p

Structural Insights into Human Parainfluenza Virus 3 Hemagglutinin–Neuraminidase Using Unsaturated 3‑<i>N</i>‑Substituted Sialic Acids as Probes

A novel approach to human parainfluenza virus 3 (hPIV-3) inhibitor design has been evaluated by targeting an unexplored pocket within the active site region of the hemagglutinin–neuraminidase (HN) of the virus that is normally occluded upon ligand engagement. To explore this opportunity, we developed a highly efficient route to introduce nitrogen-based functionalities at the naturally unsubstituted C-3 position on the neuraminidase inhibitor template <i>N</i>-acyl-2,3-dehydro-2-deoxy-neuraminic acid (<i>N</i>-acyl-Neu2en), via a regioselective 2,3-bromoazidation. Introduction of triazole substituents at C-3 on this template provided compounds with low micromolar inhibition of hPIV-3 HN neuraminidase activity, with the most potent having 48-fold improved potency over the corresponding C-3 unsubstituted analogue. However, the C-3-triazole <i>N</i>-acyl-Neu2en derivatives were significantly less active against the hemagglutinin function of the virus, with high micromolar IC₅₀ values determined, and showed insignificant <i>in vitro</i> antiviral activity. Given the different pH optima of the HN protein’s neuraminidase (acidic pH) and hemagglutinin (neutral pH) functions, the influence of pH on inhibitor binding was examined using X-ray crystallography and STD NMR spectroscopy, providing novel insights into the multifunctionality of hPIV-3 HN. While the 3-phenyltriazole-<i>N</i>-isobutyryl-Neu2en derivative could bind HN at pH 4.6, suitable for neuraminidase inhibition, at neutral pH binding of the inhibitor was substantially reduced. Importantly, this study clearly demonstrates for the first time that potent inhibition of HN neuraminidase activity is not necessarily directly correlated with a strong antiviral activity, and suggests that strong inhibition of the hemagglutinin function of hPIV HN is crucial for potent antiviral activity. This highlights the importance of designing hPIV inhibitors that primarily target the receptor-binding function of hPIV HN

X-ray Absorption Near Edge Structure analysis of endogenously expressed CorA.

<p>XANES spectrum of crystalline CorA, recorded in steps of 0.7-edge feature at 8984 eV, typical for Cu(I) compounds (marked with an arrowhead).</p