Solution NMR structure of human integrin α1-TMC in LDAO micelles.

<p>(A) The backbone superposition of the final ten structures with the lowest energies. (B) Cartoon representation of the structure of integrin α1-TMC. G1152 indicates the position of the transmembrane helix kink.</p

Integrin α1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

<div><p>Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region.</p></div

Structural comparison of integrin α1 and αIIb.

<p>Structure of α1-TMC (1135–1179) and backbone structure comparisons of α1-TM(1142–1169) with αIIb-TM structures indicate different bent regions in the transmembrane helix. The PDB number for each structure is listed below. (A) Structure ensemble of integrin α1-TMC in LDAO micelles; (B) Structure ensemble of integrin α1-TM in LDAO; (C) αIIb-TM (966–993) in bicelles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Lau3" target="_blank">[22]</a>; (D) αIIb-TM from IntαIIb/β3 complex in bicelles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Lau1" target="_blank">[20]</a>; (E) αIIb-TM (966–993) from αIIb/β3 complex in organic/aqueous solvents, 50% CD₃CN/50% H₂O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Yang1" target="_blank">[19]</a>.</p

Resonance assignment and Backbone ¹⁵N relaxation analysis of integrin α1-TMC in LDAO micelles.

<p>(A) Resonance assignment of integrin α1-TMC in LDAO micelles. Site-specific analysis of backbone amide ¹⁵N longitudinal relaxation T1 (B), transverse relaxation T2 (C) and steady-state ¹H-¹⁵N NOE (D) of integrin α1-TMC in LDAO micelles.</p

Application of Site-Specific ¹⁹F Paramagnetic Relaxation Enhancement to Distinguish two Different Conformations of a Multidomain Protein

Paramagnetic relaxation enhancement (PRE) provides long-distance restraints in solution NMR protein structural studies. It has been shown previously that L27tan, a protein with tandem L27-domains, has two possible conformations. Here, ¹⁹F was site-specifically introduced to L27tan via the incorporation of an unnatural amino acid, trifluoromethyl-phenylalanine (tfmF). Different ¹⁹F signal intensity attenuations were observed at different L27tan sites, due to different distances between the site-specifically incorporated tfmF and site-directed spin radical labeling. Analysis of the ¹⁹F detection PRE showed that the L27tan protein had a closed conformation in solution. This ¹⁹F detection PRE method could be further applied in distance measurements for proteins of large size, including multidomain proteins or membrane proteins

Discovery of a Siderophore Export System Essential for Virulence of <em>Mycobacterium tuberculosis</em>

<div><p>Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an <em>Mtb</em> mutant lacking the <em>mmpS4</em> and <em>mmpS5</em> genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of <em>mmpS4</em> and <em>mmpS5</em> did not change uptake of carboxymycobactin by <em>Mtb</em>. Thin layer chromatography showed that the Δ<em>mmpS4/S5</em> mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in <em>Mtb</em> and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the <em>mmpS4/S5</em> deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type <em>Mtb</em>. This is the strongest attenuation observed so far for <em>Mtb</em> mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of <em>Mtb</em>.</p> </div

MmpS4 and MmpS5 are not involved in iron sensing or uptake of siderophores.

<p><b>A.</b> GFP fluorescence was measured in wt <i>Mtb</i> mc²6230, Δ<i>mmpS4/S5</i>, and Δ<i>mbtD</i>::<i>loxP</i> strains containing a <i>gfp</i>-based iron-regulated reporter construct. Strains were grown in 7H9 media and fluorescence was measured two days after the addition of carboxymycobactin (cMBT) (black bars) or blank control (grey bars). Experiments were performed in triplicate and are shown with standard deviations. <b>B.</b> Uptake of ⁵⁵Fe loaded cMBT by <i>Mtb</i> Δ<i>mmpS4/S5</i> (black circles) and Δ<i>mmpS4/S5</i> Δ<i>mbtD::hyg</i> (white triangles). Assays were performed at 37°C using a final concentration of 0.25 µM cMBT, 0.45 µCi ⁵⁵Fe in triplicate. Standard deviations are shown.</p

MmpS4 or MmpS5 is required for growth of <i>M. tuberculosis</i> under iron-limited conditions.

<p><b>A.</b> Expression of MmpS4 and MmpS5 in <i>Mtb</i>. Whole cell lysates from wt <i>Mtb</i> mc²6230, Δ<i>mmpS4</i>, Δ<i>mmpS5</i>, Δ<i>mmpS4/S5</i>, Δ<i>mmpS4/S5+mmpS4/S5</i>, Δ<i>mbtD</i>::<i>hyg</i>, and Δ<i>mmpS4/S5/mbtD</i>::<i>hyg</i> were probed by Western blot by using rabbit polyclonal antibodies raised against MmpS4 and MmpS5. The cytoplasmic fructose 1,6-bisphosphatase GlpX was used as a loading control and detected using an anti-GlpX antiserum. <b>B, C.</b> Serial dilutions of log-phase cultures of <i>Mtb</i> mc²6230, Δ<i>mmpS4</i>, Δ<i>mmpS5</i>, Δ<i>mmpS4/S5</i>, Δ<i>mmpS4/S5</i> fully complemented with <i>mmpS4</i> and <i>mmpS5</i>, Δ<i>mbtD</i>::<i>hyg</i>, and Δ<i>mmpS4/S5</i>Δ<i>mbtD</i>::<i>hyg</i> were spotted on low iron glycerol-alanine-salts (GAS) plates (<b>B</b>), or on low iron GAS plates with 5 µM hemoglobin as an iron source (<b>C</b>).</p

Effect of <i>mmpS4</i> and <i>mmpS5</i> on the survival of mice infected with <i>M. tuberculosis</i>.

<p>Survival of mice infected with wt <i>Mtb</i> H37Rv (ML617), Δ<i>mmpS4/S5</i> (ML618), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS5</i> (ML619), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS4</i> (ML620), or Δ<i>mmpS4/S5</i> fully complemented with <i>mmpS4</i> and <i>mmpS5</i> (ML624). Thirteen mice were infected with each strain. Mice were euthanized at day 169.</p

MmpS proteins interact with MmpL proteins.

<p><b>A.</b> Genetic interactions between MmpS4 and MmpS5 proteins and their cognate MmpL proteins. Percent of growth in iron-restricted medium (7H9 medium containing 50 µM 2,2′-dipyridyl) of triple mutants Δ<i>mmpS4/L4/S5</i> and Δ<i>mmpS4/S5/L5</i> strains and those strains complemented with <i>mmpS4</i> or <i>mmpS5</i> compared to growth in iron-rich media. <b>B.</b> Interaction of the C-terminal soluble domain of MmpS4 (residues 52–140) with the L1 loop of MmpL4 (residues 58–199) by an <i>in vitro</i> pull down assay.</p