Radioactive Gel shift assay to probe for MtbLigA-Mtbβ-Clamp interactions.

<p>P³² labelled Mtbβ-Clamp (90 nM) was titrated against increasing concentration of MtbLigA. Samples were analysed on 6% Native PAGE. Shifts were analysed by autoradiography. No interaction could be detected between the proteins.</p

Peptide binding groove of the Mtbβ-Clamp, structural alignments and inhibitor interactions.

<p>(<b>A</b>) The hydrophobic groove, depicted in <i>yellow</i>, is present between domains II and III of each protomer. The residues of the respective hydrophobic grooves were found to be quite conserved among bacterial β-clamps. (<b>B</b>) Structural alignment of the peptide binding groove from the respective crystal structures of the Mtbβ-clamp (<i>blue</i>) and <i>E. coli</i> β-clamp (<i>red</i>). The Mtbβ-Clamp residues are numbered. (<b>C</b>) Superposition of the Mtbβ-Clamp crystal structure onto that of the <i>E. coli</i> β-clamp -RU7 inhibitor complex (PDB: 3D1G). The surface of the Mtbβ-clamp is colored <i>green</i> while the peptide binding pocket of Mtbβ-Clamp is colored according to the sequence conservation between Mtbβ-clamp and <i>E. coli</i> β-clamp; <i>dark pink</i> represent identical residues while <i>light pink</i> represents homologous residues. The RU7 inhibitor is depicted in <i>cyan</i> stick representation. The Pol IV peptide (yellow, stick representation) from its crystal structure complex with the <i>E. coli</i> clamp (PDB: 3D1E) is also shown. The inhibitor mainly interacts with subsite 1 of the peptide binding site.</p

Interactions of the <i>M.tuberculosis</i> and <i>E. coli</i> β-Clamps with various proteins.

<p>Mtbβ-Clamp^OG (90 nM) was titrated with increasing concentration of the γ-clamp loader (Rv3721c) and LigA respectively (labeled in the figure). Interactions between the β-Clamp and LigA from <i>E. coli</i> was used as positive control where <i>E.coli</i> β-Clamp^OG (90 nM) was titrated with increasing concentration of <i>E.coli</i> LigA, and MtbLigA respectively. The <i>E. coli</i> LigA was also titrated against the Mtbβ-Clamp^OG to probe for their interactions too. BSA was used as a control for non-specific interactions. Changes to the relative fluorescence intensity were observed at λ_max 510.</p

Polar inter-subunit interactions in the Mtbβ-Clamp dimer.

<p>Polar inter-subunit interactions in the Mtbβ-Clamp dimer.</p

Electrostatic surface representation of the Mtbβ-Clamp and mapping of residues that interact with DNA.

<p>(<b>A</b>) <i>Red</i> indicates the negatively electrostatic potential; <i>white</i> indicates neutral and <i>blue</i> corresponds to positively charged regions. The central cavity is positively charged and is in line with its role in binding DNA. (<b>B</b>) Mapping of residues that interact with DNA in the <i>E. coli</i> β-clamp with that of corresponding ones in the Mtbβ-Clamp. Residues within 4 Å of the substrate after superposition of the respective crystal structures were identified and some of the conserved ones are labeled.</p

Interactions at the dimeric interface in the Mtbβ-Clamp.

<p>The two protomers are distinctly colored for clarity with the <i>van der Waals</i> surface overlaid on the cartoon representation (<i>top</i>). The respective interfaces are depicted in <i>cyan</i>. The close up of the two respective interfaces (<i>below left & right respectively</i>) depict the salt-bridges by <i>black</i> dotted lines. Some of the interacting residues are labeled.</p

Data collection and refinement statistics for LinA-type2.

*<p>Values in parentheses are for the highest resolution shell (3.59−3.5 Å).</p><p>R_merge = ΣhΣi|Ih, i−Ih|/ΣhΣiIh,i, where Ih is the mean intensity of the i observations of symmetry related reflections of h. R_factor = Σ|Fobs−Fcalc|/ΣFobs, where Fobs = FP, and Fcalc is the calculated protein structure factor from the atomic model. R_free was calculated using 5.0% of the data that was not used during the refinements.</p

Enantioselectivity of LinA-type2.

<p>Enantioselective transformation of (+) and (−) α-HCH by LinA-type1 & -type2 proteins respectively, analyzed using chiral capillary column gas chromatography as detailed in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050373#s4" target="_blank">Materials & methods</a></i>. The arrows indicate the position of the pentachlorocyclohexene products. Clearly the -type1 and -type2 proteins exhibit opposite enantiomer specificity.</p

Intersubunit salt-bridges in both LinA proteins.

<p>Potential salt-bridges with distance cut-off of 3.6 Å between respective atoms. Salt bridges reported in the earlier LinA-UT26 are shown in bold.</p>*<p>Additional salt bridge identified in the -type2 protein. Calculations were performed using PISA (<a href="http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html" target="_blank">http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html</a>).</p

Salt-bridge between R31’-E81 in LinA-type2.

<p>A close-up of the LinA-type1 structure superposed onto the -type2 co-ordinates to illustrate the conformational difference between the R31 and E81 residues. The -type1 protein is shown in yellow while the -type2 protein is shown in blue. G23 and Q20, two residues that are important for thermostability of the -type2 protein are depicted as red sticks for clarity.</p