Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from <i>Toxoplasma gondii</i>

<div><p>The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In <i>T</i>. <i>gondii</i> and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of <i>T</i>. <i>gondii</i> F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key F_O subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.</p></div

Identifying the dimer and monomer forms of <i>T</i>. <i>gondii</i> F-type ATP synthase by BNP analysis.

<p>(A) Mitochondria lysates were prepared from tachyzoites stage transgenic parasites expressing <i>Tg</i>ATPβ-YFP-HA protein were separated by BNP. Lane M, native molecular weight markers; lane A, Coomassie blue staining of BNP gel; lane B, western blotting of BNP gel using α-HA antibodies. The dimer and monomer forms are indicated by arrows. The boxed regions in lane A correspond to the excised gel pieces, which were processed for LC-MS/MS analysis. (B) In-gel ATPase activity assays following BNP separation confirms that the dimer and monomer forms of F-type ATP synthase are functionally intact. BNP, blue native PAGE; HA, hemagglutinin; LC-MS/MS, liquid chromatography–tandem mass spectrometry; <i>Tg</i>ATP-β, <i>T</i>. <i>gondii</i> ATP synthase β subunit; <i>Tg</i>ATP-OSCP, <i>T</i>. <i>gondii</i> ATP synthase OSCP subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Identification of novel <i>T</i>. <i>gondii</i> F-type ATP synthase subunits from LC-MS/MS analysis.

<p>(A) The Venn diagram shows shared identification of proteins following BNP, SEC, and IP sample preparation. Total number of proteins identified in each technique is given within brackets outside the Venn diagram. The numbers shown in white font and within brackets are the final set of proteins assigned as subunit components of <i>T</i>. <i>gondii</i> F-type ATP synthase. (B) List of all genes identified in this work by mass spectrometry analysis. Gene ID and product description details are from Toxodb.org (release 36). The entries shown in bold indicate that the protein was detected with high confidence from only BNP and IP samples. Asterisk indicates that the corresponding peptides were detected in only one of the replicate runs for SEC sample. BNP data are a combination of experiments done with both <i>Tg</i>ATPβ-YFP-HA–and <i>Tg</i>ATPOSCP-YFP-HA–expressing transgenic parasites. SEC and IP data are from <i>Tg</i>ATPβ-YFP-HA and <i>Tg</i>ATPOSCP-YFP-HA expressing transgenic parasites, respectively. BNP, blue native PAGE; IP, immunoprecipitation; LC-MS/MS, liquid chromatography–tandem mass spectrometry; OSCP, oligomycin sensitivity–conferring protein; SEC, size exclusion chromatography; <i>Tg</i>ATPβ, <i>T</i>. <i>gondii</i> ATP synthase β subunit; <i>Tg</i>ATPOSCP, <i>T</i>. <i>gondii</i> ATP synthase OSCP subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Phylogenetic profile of the alveolate infrakingdom for all <i>T</i>. <i>gondii</i> F-type ATP synthase subunits.

<p>Alveolate clades are highlighted with a gray background, and their expected phylogenetic relationship is indicated by a tree structure above. Gray and white boxes indicate the presence and absence of the corresponding ortholog, respectively. The hatched boxes represent the presence of the ortholog in <i>C</i>. <i>muris</i> only and absence in <i>C</i>. <i>parvum</i> and <i>C</i>. <i>hominis</i>. The table on the left lists the gene ID for all ASAPs, along with their annotation, essentiality phenotypes (phenotype score from CRISPR/Cas9 knockout study [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref050" target="_blank">50</a>]), and protein localization. The key for localization annotation is given below the table. ASAP, ATP synthase–associated protein; Cas9, CRISPR-associated 9; CRISPR, clustered regularly interspaced short palindromic repeat; OSCP, oligomycin sensitivity–conferring protein.</p

Detail of F-type ATP synthase subunits missing in <i>T</i>. <i>gondii</i> and other apicomplexan parasites.

<p>(A) Schematic representation of core subunit composition of F-type ATP synthase. The different subunits are color coded and annotated. Middle, the complete set of subunits of the core enzyme; left and right, subunits identified and not identified by in silico methods in apicomplexan genome. (B) Table showing the presence/absence of orthologs of yeast and bovine F-type ATP synthase in <i>Apicomplexa</i> and other alveolate species. +/−, presence/absence of ortholog; blue, diverged (no clear orthologs) functional equivalent; light blue, experimentally identified species-specific novel subunits (numeric values indicate the total number of such novel subunits). Species names: <i>Pfal</i>, <i>P</i>. <i>falciparum</i>; <i>Pviv</i>, <i>Plasmodium vivax</i>; <i>Tpar</i>, <i>Theileria parva</i>; <i>Bbov</i>, <i>Babesia bovis</i>; <i>Tgon</i>, <i>T</i>. <i>gondii</i>; <i>Ncan</i>, <i>Neospora caninum</i>; <i>Cmur</i>, <i>Cryptosporidium muris</i>; <i>Cpar</i>, <i>Cryptosporidium parvum</i>; <i>Chom</i>, <i>Cryptosporidium hominis</i>; <i>Cvel</i>, <i>Chromera velia</i>; <i>Pmar</i>, <i>Perkinsus marinus</i>; <i>Tthe</i>, <i>T</i>. <i>thermophila</i>; <i>Scer</i>, <i>Saccharomyces cerevisiae</i>; <i>Btau</i>, <i>Bos taurus</i>; <i>Atha</i>, <i>Arabidopsis thaliana</i>; <i>Crei</i>, <i>Chlamydomonas reinhardtii</i>; <i>Tbru</i>, <i>T</i>. <i>brucei</i>. IF1, inhibitory factor 1; OSCP, oligomycin sensitivity–conferring protein.</p

HHPred identification of novel F_O subunit a based on conserved structural features.

<p>(A) Representation of the pairwise sequence alignments generated by HHPred [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref046" target="_blank">46</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref047" target="_blank">47</a>] for the putative F_O subunit a from <i>T</i>. <i>gondii</i> and 4 other F_O subunit a proteins for which the structure is known. The table provides details of the amino acid length and a probability score for the prediction from the hit alignments. The red lines indicated the 3 transmembrane domains present in the <i>T</i>. <i>gondii</i> protein. (B) Protein sequence alignments and secondary structure information were made using the Clustal Omega [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref048" target="_blank">48</a>] and ESPript [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref049" target="_blank">49</a>] software, and only the C-terminal portion of respective proteins is shown. The names of alveolate species are highlighted in blue. For the nonalveolate species included in the alignment, the F_O subunit a is either readily identified from sequence (<i>Scer</i>, <i>Hsap</i>, <i>Atha</i>, <i>Ecol</i>, and <i>Pten</i>) or has been experimentally determined (<i>Crei</i> and <i>Poly</i>). Positions with similar amino acids are highlighted in red in the alignment. The arginine and glutamine residues, highlighted in yellow, are conserved in all species and important for function. The helices (α9, α10, and α12) shown are from the structure of <i>Poly</i> F-type ATP synthase. Species names: <i>Tgon</i>, <i>T</i>. <i>gondii</i>; <i>Hham</i>, <i>Hammondia hammondi</i>; <i>Pfal</i>, <i>P</i>. <i>falciparum</i>; <i>Pviv</i>, <i>P</i>. <i>vivax</i>; <i>Tann</i>, <i>Theileria annulata</i>; <i>Bbov</i>, <i>B</i>. <i>bovis</i>; <i>Cmur</i>, <i>C</i>. <i>muris</i>; <i>Cvel</i>, <i>C</i>. <i>velia</i>; <i>Crei</i>, <i>C</i>. <i>reinhardtii</i>; <i>Poly</i>, <i>Polytomella</i> sp.; <i>Scer</i>, <i>S</i>. <i>cerevisiae</i>; <i>Hsap</i>, <i>Homo sapiens</i>; <i>Atha</i>, <i>A</i>. <i>thaliana</i>; <i>Ecol</i>, <i>Escherichia coli</i>; <i>Pden</i>, <i>Paracoccus denitrificans</i>.</p

Partial purification of dimer and monomer forms of <i>T</i>. <i>gondii</i> F-type ATP synthase by chromatography.

<p>(A) Ion exchange (DEAE sepharose) separation of mitochondrial lysates prepared from <i>Tg</i>ATPβ-YFP-HA expressing transgenic parasites. Absorbance at 280 nm (filled circles) and NaCl concentration (open circles) are plotted for each fraction. Fractions 5 and 18 are marked with arrows. Bottom panel shows SDS-PAGE western blotting for fractions 5–18 to find out the elution profile of <i>Tg</i>ATPβ-YFP-HA. (B) Size exclusion profile of the pooled fractions from ion exchange chromatography. The absorbance at 280 nm is plotted for each fraction. The size exclusion column was calibrated with the following native markers: thyroglobulin (labeled “T,” 660 kDa), ferritin (labeled “F,” 440 kDa), conalbumin (labeled “C,” 75 kDa), Ovalbumin (labeled “O,” 45 kDa). Peak elution volume for each marker is indicated by arrow. Fractions 1–3, 4–6, and 7–9 were pooled, concentrated, and subject to SDS-PAGE western blotting to detect <i>Tg</i>ATPβ-YFP-HA, as shown in bottom panel. DEAE, diethylaminoethanol; OD, optical density; <i>Tg</i>ATPβ, <i>T</i>. <i>gondii</i> ATP synthase β subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Efforts To Access the Potent Antitrypanosomal Marine Natural Product Janadolide: Synthesis of Des-<i>tert</i>-butyl Janadolide and Its Biological Evaluation

To identify novel antitrypanosomal agents based on Janadolide, a potent macrocyclic polyketide–peptide hybrid, a macrolactonization strategy was explored. We prepared des-<i>tert</i>-butyl Janadolide and evaluated its antitrypanosomal activity. Our findings suggest that the <i>tert</i>-butyl group is necessary for the desired bioactivity

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Specific Stereoisomeric Conformations Determine the Drug Potency of Cladosporin Scaffold against Malarial Parasite

The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral center antimalarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme-, and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency whereas changes at C3 are sensed by rotameric flipping of glutamate 332. Given that scores of antimalarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of antimicrobial drug development