Structural similarity of p34ct compared to other vWA domains.

<p>Superposition of the p34ct 1-277 structure (yellow) with (A) the human Integrin α1 I domain (pdb entry 1PT6), (B) the human vWF A1 domain (pdb entry 1AUQ) and (C) Rpn10 from <i>Schizosaccharomyces pombe</i> (pdb entry 2×5N, each in cyan). The putative metal coordination site in p34ct is enlarged (D) and compared to the MIDAS motif in human Integrin α1 (cyan), with the 5 MIDAS elements (A1 – A5) shown in stick representation and the bound Mg²⁺ ion in Integrin α1 (1PT6) depicted as a green sphere. Residues corresponding to A1 – A5 are depicted next to the structural models.</p

Multiple sequence alignment of p34 proteins from five different organisms.

<p>The alignment was obtained with MUSCLE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102389#pone.0102389-Edgar1" target="_blank">[56]</a> and visualized via JalView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102389#pone.0102389-Waterhouse1" target="_blank">[57]</a> after manual modification. Regions visible in the p34ct vWA structure are boxed in green and secondary structure elements are indicated above the sequence, with arrows representing β-strands while coils are used for α-helices. Conserved residues between species are colored in different shades of blue, depending on the degree of conservation. The N-C-Linker present in <i>C. thermophilum</i>, but absent in <i>S. cerevisiae</i>, <i>M. musculus</i>, human and <i>A. thaliana</i> p34, is highlighted in red. The highly conserved C-terminal C4 zinc finger motif is indicated in orange.</p

DNA binding properties and oligomeric state of p34ct samples.

<p>SDS-PAGE of purified samples after size-exclusion chromatography, with the position of each p34ct construct indicated by red arrows (A). For DNA binding assays (B) the samples were separated on native agarose gels and the DNA visualized via Midori Green staining. Neither p34ct (B, left) nor p34ct 1–277 (not shown) were able to bind single stranded (ss) or double stranded (ds) DNA. DNA Polymerase I from <i>Bacillus caldotenax</i> served as a positive control for DNA binding (B, right). The multi angle light scattering analysis of p34ct (C) and p34ct 1–277 (D) samples was coupled to size-exclusion chromatography. The sample concentration in mg/ml as a function of the differential refractive index (dRI) is shown in red whereas the calculated molar mass is indicated as a scatter plot, with one data point per measurement and second (C and D).</p

Potential binding sites for the TFIIH p44 subunit.

<p>(A) A structure of the human C-terminal domain of p44 (1Z60) is shown as a cartoon model and with the electrostatic potential (contoured at ± 3.0 k_bT/e_c) mapped onto its surface. The zinc ions bound to the C4C4 motif in p44 are shown in grey and the two regions most likely involved in p34 binding are circled in red. (B) The electrostatic potential contoured at ± 3.0 k_bT/e_c is mapped onto the p34ct surface (A) in blue (positive), red (negative) and white (neutral) and suggests two putative binding sites for the p44ct C4C4 domain (circled in cyan). (C) The conservation of surface residues in the p34ct vWA domain is depicted, with the different shades of blue reflecting the variable degree of conservation, in analogy to the color code used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102389#pone-0102389-g002" target="_blank">Figure 2</a>.</p

Catabolism of the Cholesterol Side Chain in <i>Mycobacterium tuberculosis</i> Is Controlled by a Redox-Sensitive Thiol Switch

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis (TB), is a highly successful human pathogen and has infected approximately one-third of the world’s population. Multiple drug resistant (MDR) and extensively drug resistant (XDR) TB strains and coinfection with HIV have increased the challenges of successfully treating this disease pandemic. The metabolism of host cholesterol by <i>Mtb</i> is an important factor for both its virulence and pathogenesis. In <i>Mtb</i>, the cholesterol side chain is degraded through multiple cycles of β-oxidation and FadA5 (Rv3546) catalyzes side chain thiolysis in the first two cycles. Moreover, FadA5 is important during the chronic stage of infection in a mouse model of <i>Mtb</i> infection. Here, we report the redox control of FadA5 catalytic activity that results from reversible disulfide bond formation between Cys59-Cys91 and Cys93-Cys377. Cys93 is the thiolytic nucleophile, and Cys377 is the general acid catalyst for cleavage of the β-keto-acyl-CoA substrate. The disulfide bond formed between the two catalytic residues Cys93 and Cys377 blocks catalysis. The formation of the disulfide bonds is accompanied by a large domain swap at the FadA5 dimer interface that serves to bring Cys93 and Cys377 in close proximity for disulfide bond formation. The catalytic activity of FadA5 has a midpoint potential of −220 mV, which is close to the <i>Mtb</i> mycothiol potential in the activated macrophage. The redox profile of FadA5 suggests that FadA5 is fully active when <i>Mtb</i> resides in the unactivated macrophage to maximize flux into cholesterol catabolism. Upon activation of the macrophage, the oxidative shift in the mycothiol potential will decrease the thiolytic activity by 50%. Thus, the FadA5 midpoint potential is poised to rapidly restrict cholesterol side chain degradation in response to oxidative stress from the host macrophage environment

DNA Helicases used in this study.

<p>DNA Helicases used in this study.</p

Biochemical parameters of ctXPD and variants.

<p>Biochemical parameters of ctXPD and variants.</p

Sequestration of FANCJ, but not RECQ1, by cyclo dA.

<p><i>A,</i> Schematic of sequestration assay. Sequestration assays were performed with 9.6 nM FANCJ or 8.8 nM RECQ1 and the indicated concentrations of the competitor DNA forked duplex at 30°C (FANCJ) or 37°C (RECQ1) under sequestration assay conditions described in the Materials and Methods. <i>B</i> and <i>C,</i> FANCJ (<i>B</i>) or RECQ1 (<i>C</i>) unwinding of undamaged 19 bp tracker DNA substrate after incubation with unlabeled forked duplex DNA molecules that contained cyclo dA in the top, bottom, or neither strand. <i>D and E,</i> Quantification of FANCJ and RECQ1 helicase activity from representative sequestration experiments shown in panels <i>B</i> and <i>C</i>, respectively.</p

Effect of a site- and strand-specific cyclopurine lesion on BLM or RECQ1 helicase activity.

<p>Helicase reactions were carried out by incubating the indicated BLM or RECQ1 concentrations with 0.5 nM forked duplex DNA that contained a cyclopurine lesion in the top strand (nontranslocating-Cyclo T), bottom strand (translocating-Cyclo B), or neither strand (Control) at 37°C for 15 min (RECQ1) or 30 min (BLM) under standard helicase assay conditions described in the Materials and Methods. <i>A,</i> BLM unwinding of undamaged and cyclo dA damaged DNA substrates. Lane 1, heat-denatured DNA substrate control; lane 2, no enzyme control; lanes 3–7, indicated concentrations of BLM. <i>B,</i> Quantification of BLM helicase activity on cdA substrates with error bars. <i>C,</i> BLM unwinding of undamaged and cyclo dG damaged DNA substrates. Lane 1, no enzyme control; lanes 2–6, indicated concentrations of BLM, lane 7 heat-denatured DNA substrate control. <i>D,</i> Quantification of BLM helicase activity on cdG substrates with error bars. <i>E,</i> RECQ1 unwinding of undamaged and cyclo dA damaged DNA substrates. Lane 1, no enzyme control; lanes 2–9, indicated concentrations of RECQ1; lane 10, heat-denatured DNA substrate control. <i>F,</i> Quantification of RECQ1 helicase activity on cdA substrates with error bars. <i>G,</i> RECQ1 unwinding of undamaged and cyclo dG damaged DNA substrates. Lane 1, no enzyme control; lanes 2–9, indicated concentrations of RECQ1; lane 10, heat-denatured DNA substrate control. <i>H,</i> Quantification of RECQ1 helicase activity on cdG substrates with error bars.</p

NER activity of hsXPD and its variants.

<p>(a) Purified core-TFIIH (rIIH6) resolved by SDS-PAGE followed by Coomassie staining and Western blot analysis. (b and c) NER activity of reconstituted TFIIH containing mutant XPD proteins. XPD wild type or variants (100 and 200 ng) were mixed with purified core-TFIIH (rIIH6) and added to an <i>in vitro</i> double-incision assay using recombinant NER factors. The reaction was analyzed by electrophoresis followed by autoradiography. Incision activities from three independent experiments were quantified and normalized to wild type. (d and e) Host cell reactivation activity of XPD variants. HD2 fibroblasts were transfected with a reporter plasmid expressing firefly luciferase previously exposed to 1,000 J/cm² UVC-light (254 nm) (d) or with the nonirradiated control (e) in combination with vector expressing renilla luciferase to normalize transfection efficiencies and pIERS2-EGFP expressing XPD wild-type or mutant proteins. The firefly luciferase activity in cell lysates (48 h posttransfection), normalized with the internal renilla luciferase standard, assesses repair complementation. The values of three independent experiments are presented as percentages, with 100% being the level of luciferase activity obtained with wild-type XPD. The data used to generate panels (c), (d), and (e) have been deposited as supplementary information in xls format (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001954#pbio.1001954.s007" target="_blank">Table S3</a>).</p