The oligomeric interface.

<p>The interaction between Lys-4 of one monomer (in blue) and the neighboring N-Lsr2 dimer (in yellow and pink). This interaction is present in both crystal forms. Polar interactions are labelled with pink dotted lines and selected residues are labeled. An electron density map contoured at 1σ is shown as blue mesh.</p

Cartoon diagrams of the Lsr2 N-terminal domain in the <i>P</i>2₁ crystal structure.

<p>(A) A depiction of the monomer: the α-helix, β-strands and chain termini are labeled and the chain is colored blue-red (N-terminus to C-terminus); (B) The dimer as seen in the crystal structure, one chain is colored and the other is grey; (C) A view orthogonal to that of B showing residues critical for dimerization; (D) Lsr2 N-terminal domain oligmerization as generated by crystallographic symmetry. The N-terminus of one dimer donates one strand forming an anti-parallel β-sheet. The second strand is presented by a neighboring dimer. The β-sheet linking two dimers is shown as β0; (E) Crystallographic symmetry (in space group <i>P</i>2₁) showing the unit cell (in green) projected perpendicular to the b-axis. The two-fold screw axis generates alternating Lsr2 chains that lie back-to-back along the horizontal (in this view). For all figures protein depictions were drawn with PYMOL.</p

Addition of trypsin to N-Lsr2 facilitates oligomerization.

<p>(A) Size exclusion chromatogram of freshly purified Lsr2 N-terminal domain showing a single dimer peak; (B) A chromatogram of the same protein construct used for crystallography (now 14 wks old) demonstrating larger oligomeric forms in solution; (C) The addition of trypsin (1∶500) for 5 min at room temperature to a fresh sample of Lsr2 N-terminal domain accelerates the formation of large oligomeric species in solution. SDS-PAGE gels showing relevant fractions from the purification: L  =  protein loaded on the column; A, B and C represent protein from fractions as labeled on the chromatogram. The N-terminal domain runs at a higher M_r than expected on SDS-PAGE gels due to the presence of a 6xHis-tag plus a linker.</p

A model for chromosomal DNA organization suggested by hexagonal crystal packing.

<p>(A) The N-terminal Lsr2 dimerization domain in the <i>P</i>3₁21 space group in two orientations; (B) Proposed orientation of DNA (in blue) between N-terminal chains and a 90° rotation illustrating this from above. The C-terminal DNA-binding domains (in orange) have been positioned according to Gordon <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038542#pone.0038542-Gordon3" target="_blank">[22]</a>; (C) Our model of full-length Lsr2 binding to DNA and forming chains cross-linking multiple strands of DNA. The dimension at the bottom of the figure is consistent with the compact fibrils seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038542#pone-0038542-g006" target="_blank">Figure 6B</a>.</p

The C-terminal boundaries of Lsr2 N-terminal domain constructs.

<p>The C-terminal boundaries of Lsr2 N-terminal domain constructs.</p

Conserved amino acids for the N-terminal domain of Lsr2.

<p>(A) Relative conservation of amino acids at different positions from a multiple sequence alignment are depicted using HMM Logo (<a href="http://pfam.sanger.ac.uk/family/PF11774.2" target="_blank">http://pfam.sanger.ac.uk/family/PF11774.2</a>). The height of the letters represents the relative entropy and the width represents the relative contribution of the position to the overall protein family. The pink bars represent regions of insertion in the alignment. Amino acid residue colours reflect their biological propertes (red  =  charged; blues  =  polar, uncharged; yellows  =  aliphatic; greens  =  aromatic). The amino acid sequence for Lsr2 from <i>M. tuberculosis</i> is shown across the top of the HMM logo for comparison and residues that are well-conserved are in bold. (B) Cartoon diagram of Lsr2 N-terminal dimerization domain showing conserved residues. The structure is for the <i>P</i>2₁ crystal form. Conserved residues for each chain are shown in yellow and orange respectively. Yellow residues from chain A are labeled. The chain termini are also labeled.</p

Negatively stained Lsr2/DNA complexes visualized by transmission electron microscopy show large morphological changes upon trypsin treatment.

<p>(A) Lsr2 co-purified with <i>E. coli</i> genomic DNA; (B) Lsr2 after trypsin digestion (1∶500 ratio) for 30 minutes; Arrows A and B point to condensed structures commonly seen during the trypsin digestion time series. (Scale bars  = 100 nm); (C) A cartoon representation of protein oligomerization and DNA compaction after trypsin digestion. Lsr2 N-terminal dimerization domain is depicted in green and the C-terminal DNA-binding domain is depicted in orange. The downward pointing arrows correlate the features in (B) with their cartoon representations in (C).</p

Trypsin digestion of full-length Lsr2 co-purified with <i>E. coli</i> genomic DNA.

<p>Trypsin (1∶500) initiates the compaction of co-purified DNA by Lsr2 over 1 h as visualised on a 1% agarose gel (SYBR® safe DNA stain). Subsequent digestion of the compacted DNA using DNase shows protection of the DNA by Lsr2. Equivalent samples were run on an SDS-PAGE gel to show the presence of Lsr2 (both monomer and dimer forms) in the sample after trypsin digestion. Components are labeled: C  =  condensation of DNA upon treatment with trypsin; P  =  protection of DNA; D  =  Lsr2 dimer; M  =  Lsr2 monomer).</p

Overall structure of the N-terminal domain of A46.

<p>A, Two views of the structure of A46(1–83). The dimer comprises subunit A (7 β-strands) and subunit B (6 β-strands) with the subunits coloured as rainbows from the N- to the C-termini. Myristic acid co-crystallized as a ligand inside of the cavity and is depicted as sticks; carbon atoms are blue and oxygen ones are red. B, Superimposition of subunit B on subunit A of A46(1–83) dimer. Subunit A (in blue) and subunit B (in red) were superimposed in PyMOL. MYR, myristic acid. The amino acids most differing within subunits are indicated. Panel B is related to panel A by a counter-clockwise rotation of 30°. Drawings were made using PyMOL [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref029" target="_blank">29</a>].</p

Inhibitory effect of full-length and truncated constructs of A46 on the IL-1β stimulated NK-κB transcription.

<p>HEK293T cells were transfected with the indicated amounts of plasmids. Plasmid amounts were adjusted so that approximately the same amounts of each A46 variant were expressed. After 40 h of incubation, cells were stimulated with 0.32 ng/ml of IL-1β and incubated for a further 6 h. NF-κB reporter gene activity was then measured. Data are expressed as the relative stimulation from a representative experiment from a minimum of three separate experiments, each performed in triplicate. Error bars represent the standard deviation from the mean. EV, empty vector.</p