Parasite morphology and global protein phosphorylation pattern of PKG inhibitor-treated <i>P. falciparum</i> schizonts.

<p>(<b>A</b>) Immunofluorescent staining of DMSO/compound 1-treated WT schizonts using antibodies detecting (i) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>, (ii) PfSUB1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoh1" target="_blank">[14]</a> and (iii) PfAMA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Harris1" target="_blank">[23]</a>. Representative images are shown for each staining together with parasite nuclei stained with DAPI. Bars ∼5 µM. (<b>B</b>) Metabolic labelling of phosphoproteins in <i>P. falciparum</i> schizonts. Autoradiographs of (i) 3D7 WT and (ii) gatekeeper mutant 3D7 PfPKG_T618Q schizonts, treated with ³²P-orthophosphate and DMSO (−) or compound 2 (+) prior to lysis, ÄKTA anion exchange chromatography (fractions 10–14 are shown) and separation by SDS-PAGE. Rectangular boxes highlight bands that show a differential signal following inhibitor-treatment in WT, but not PfPKG_T618Q schizonts.</p

Subcellular location of PfPKG in mature schizonts.

<p>Dual immunofluorescent detection of PfPKG-HA in fixed smears of early and late schizonts of the PfPKG-HA-3A clone together with (<b>A</b>) PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a>, (<b>B</b>) PfBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-vanDooren1" target="_blank">[32]</a>, (<b>C</b>) PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, (<b>D</b>) PfRab11A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-AgopNersesian1" target="_blank">[33]</a> and (<b>E</b>) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>. Representative images are shown for each antibody, together with bright field images (first column) and parasite nuclei stained with DAPI (in the merged image). Bars ∼5 µM. To quantify co-localisation, Pearson coefficients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Manders1" target="_blank">[36]</a> of the individual stains were calculated using Imaris image analysis software (Bitplane).</p

PfPKG expression peaks in late blood stages and is carbonate-soluble.

<p>(<b>A</b>) Western blots of synchronised cultures of the PfPKG-HA-3A clone and WT parasites (3D7 clone), 24 hours (mostly mid trophozoites), 30 hours (mostly late trophozoites), 41 hours (mostly early schizonts) and 46 hours (mostly late schizonts) post invasion were detected with anti-HA and anti-humanPKG, respectively. Blots were re-probed with an antibody against Pfαtubulin to estimate the relative total protein loading between lanes. (<b>B</b>) Sequential solubilisation of parasite proteins from saponin-released late trophozoites and schizonts. S1: soluble protein fraction (5 mM Tris-HCl, freeze thaw); S2: peripheral membrane fraction (extraction with 100 mM Na₂CO₃); S3: integral membrane fraction (extraction with 4% SDS/0.5% TX-114/0.5×PBS). Equal volumes of the three supernatants were analysed by SDS-PAGE and Western blots were probed for the integral membrane protein PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, stripped and re-probed simultaneously for PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a> and PfPKG-HA. Densitometric analysis of the scan of the blot presented revealed that 89.3% of PfPKG-HA is present in fraction S1, while fractions S2 and S3 contain 9.5% and 1.2%, respectively. (<b>C</b>) Immunofluorescent anti-HA detection in fixed smears of erythrocytic stages of the PfPKG-HA-3A clone. Representative images of (i) a ring stage parasite, (ii) three early trophozoites, (iii) an early schizont, (iv, v) late schizonts (approximate hours post invasion: (i) 4–10, (ii) 20–26, (iii) 33–39, (iv, v) 45–48) and (vi) a stage III gametocyte are shown together with bright field images (first column) and parasite nuclei stained with DAPI (second column). Bars ∼5 µM.</p

Domain organization and overall structures of <i>Pf</i>CNB-D.

<p>(A) Domain organization of <i>Pf</i>PKG and sequence alignment between <i>Pf</i>CNB-D and <i>Hs</i>CNB-B (Human PKG I). Identical residues are highlighted in yellow and the capping residues in both proteins are highlighted in red. The capping triad residues are also marked with arrows. (B) cGMP and cAMP affinities of <i>Pf</i>CNB domains. Competition FP curves for cGMP are shown on the left and EC₅₀ values on the right. (C) Overall structure of <i>Pf</i>CNB-D without cGMP. The secondary structure elements are labeled. The phosphate binding cassette (PBC) is colored in yellow, the αB and αC helices in light cyan and blue, the N-terminal helices in light green and the β-barrel in gray. The N- and C-termini are labeled with their corresponding residue number seen in the final model. The sulfate ion co-crystallized with the protein is colored with its sulfur in yellow and oxygen in red. (D) Overall structure of the <i>Pf</i>CNB-D:cGMP complex. The structure is shown with the same color scheme as above except for cGMP. The cGMP is colored by atom type (carbon, white; nitrogen, blue; oxygen, red; and phosphorus, orange). All structure images were generated using <i>PyMOL</i> (Delano Scientific).</p

Data and refinement statistics.

<p>Data and refinement statistics.</p

Structural comparison between the apo- and cGMP bound <i>Pf</i>CNB-D.

<p>The apo and <i>Pf</i>CNB-D:cGMP complex structures are aligned at the β-barrel region (not colored). The helical subdomain of the apo structure is colored in light cyan and that of the cGMP complex structure in yellow.</p

Structural comparison between <i>Pf</i>CNB-D and CNB-B and cGMP binding pocket of <i>Pf</i>CNB-D.

<p>(A) The cGMP pockets <i>Pf</i>CNB-D and CNB-B from human PKG Iβ (PDB code: 4KU7) are shown. The cGMP pocket of <i>Pf</i>CNB-D is colored in yellow (left) and the pocket of PKG Iβ CNB-B in gray (right). Key residues that stabilize the C-helix including the capping residues are shown with transparent surface in the following color theme: side chain carbon, black; oxygen, red; nitrogen. A water molecule captured between E483, R484, and Q532 is shown as a blue sphere. The C atoms of glycine residues located between at the αB and αC helices are shown as black spheres. Hydrogen bonds are shown as dotted lines. (B) Detailed interactions between <i>Pf</i>CNB-D and cGMP. Zoomed in views for each cGMP binding site are shown on either side. The backbone amide of A485 is marked with a blue dot. The individual cGMP interacting residues are shown with the following color theme: side chain carbon, black; oxygen, red; nitrogen, blue. The residues binds cGMP with VDW contacts including the capping residues are shown with transparent surface. Hydrogen bonds are shown as dotted lines with their distances in Å units.</p

The patient with carcinoma of tongue and his nursing case report

Additional file 2: Table S2. Insertions (INS) and deletions (DEL) identified in the three lines 3D7A, A4 and F12

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