Polar inter-chain interactions between L3, αA and N-terminal regions in homology models of both enzyme variants.

<p>N-terminal region consists of residues 581–620 (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.g001" target="_blank">Fig 1A</a>).</p><p>Polar inter-chain interactions between L3, αA and N-terminal regions in homology models of both enzyme variants.</p

Polar interactions at the dimer interface of <i>Pf</i>CCT MΔK^WT and <i>Pf</i>CCT MΔK^R681H involving ⁶⁸¹RWVD⁶⁸⁴.

<p>A) Direct interactions harbouring ⁶⁸¹RWVD⁶⁸⁴ signature sequence motif in <i>Pf</i>CCT MΔK^WT. The residues involved in the inter-chain interaction are shown in stick representation, and interactions are indicated by pink dashed lines. Characteristic dimer interface distances d(R681, CA - H679’, O) and d(I680, CA - I680’, N) are denoted by blue double-headed arrows. Residues in chain B are marked with apostrophes. B) Direct interactions harbouring the ⁶⁸¹HWVD⁶⁸⁴ mutated signature sequence motif <i>Pf</i>CCT MΔK^R681H. The residues involved in the inter-chain interaction are shown in stick representation, and interactions are indicated by pink dashed lines. Characteristic dimer interface distances d(H681, CA - H679’, O) and d(I680, CA - I680’, N) are denoted by blue double-headed arrows. Residues in chain B are marked with apostrophes.</p

Inter-subunit interaction energies during molecular dynamics simulations.

<p>Interaction energies are calculated as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.e001" target="_blank">Eq 1</a> and represented by black line in case of <i>Pf</i>CCT MΔK^WT and by grey line in case of <i>Pf</i>CCT MΔK^R681H, respectively.</p

Mass spectra of <i>Pf</i>CCT MΔK^WT and <i>Pf</i>CCT MΔK^R681H proteins under native electrospray conditions.

<p>M and D indicate signals contributing monomers and dimers, respectively, while numbers denote the charge states. A) Mass spectrum of <i>Pf</i>CCT MΔK^WT measured in the present study for direct comparison (cf. also [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.ref010" target="_blank">10</a>]). B) Mass spectrum of <i>Pf</i>CCT MΔK^R681H. In the inset the 10-times enlarged graph of dimer regions (3150–4000 m/z) is shown.</p

Steady-state kinetic analysis of <i>Pf</i>CCT MΔK^WT and <i>Pf</i>CCT MΔK^R681H.

<p>A) CTP titration of the activity of the CCTs at a fixed ChoP concentration of 5 mM. The plot shows one representative experiment. Titration data are fitted with the Michaelis–Menten kinetic model assuming no cooperativity. B) ChoP titration of the activity of the CCTs at a fixed CTP concentration of 1 mM. The plot shows one representative experiment. Titration data are fitted with a kinetic model assuming substrate inhibition without cooperativity. Note the substrate inhibition effect of ChoP as an initial rate decrease is observed at higher substrate concentrations.</p

Inter-subunit interaction distances during molecular dynamics simulations.

<p>A) Variation of the atomic distance d(R/H681, CA - H679’, O) as a characteristic proximal inter-subunit contact of the catalytic domains (cf. Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.g002" target="_blank">2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.g002" target="_blank">2B</a>). Distances are represented by black line in case of <i>Pf</i>CCT MΔK^WT and by grey line in case of <i>Pf</i>CCT MΔK^R681H, respectively. B) Variation of the atomic distance d(I680, CA - I680’, N) as a characteristic proximal inter-subunit distance of the catalytic domains (cf. Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.g002" target="_blank">2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.g002" target="_blank">2B</a>). Distances are represented by black line in case of <i>Pf</i>CCT MΔK^WT and by grey line in case of <i>Pf</i>CCT MΔK^R681H.</p

Protein structure prediction of <i>Pf</i>CCT MΔK^WT and <i>Pf</i>CCT MΔK^R681H.

<p>A) Alignment of rat CCT (PDB ID: 4MVC) and <i>Pf</i>CCT MΔK^WT sequences used for modeling and MD stimulations. Numbering is according to <i>Pf</i>CCT MΔK^WT. Secondary structure elements are represented by squiggles (α-helices), arrows (β-strands) and lines (turns). In the aligned sequences, red box with white character indicates strict identity and red character means similarity in groups. R681 corresponding to R140 in rat CCT is indicated by a green star. Hydropathy (pink—hydrophobic, grey—intermediate, cyan—hydrophilic) and accessibility (blue—accessible, cyan—intermediate, white—buried) are also presented below the sequences. The layout with secondary structure elements was generated with ESPript 3.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129632#pone.0129632.ref066" target="_blank">66</a>], supplemented with visual inspection of structures. B) Conservation of the RYVD signature sequence in CCTs, shown by Weblogo. Numbering is according to the <i>Plasmodium falciparum</i> sequence, where the ⁶⁸¹RWVD⁶⁸⁴ corresponds to the ¹⁴⁰RYVD¹⁴³ in the rat sequence. C) Dimer structure of MΔK^WT homology model. Chain A is coloured in red and chain B is coloured in yellow. Important secondary structure elements are indicated.</p

Overall effective interaction energy (Winteq) and the contribution of non-bonded van der Waals (EvdWeq) and Coulomb (ECoueq) interaction energy terms and of the solvation free energy change upon dimerization (ΔGsolveq) averaged over all frames of the equilibrated phase of the productive MD simulations.

<p>The superscript ‘eq’ denotes the equilibrated phase (final 1.75 ns) of the productive MD simulations. Data from the last frame of the simulations are denoted as ‘lf’. Important geometric data such as volume ((</p><p></p><p></p><p></p><p><mi>V</mi></p><p><mi>l</mi><mi>f</mi></p><p></p><p></p><p></p><p></p>) and surface area (<p></p><p></p><p></p><p><mi>A</mi></p><p><mi>l</mi><mi>f</mi></p><p></p><p></p><p></p><p></p>), interaction surface area (<p></p><p></p><p></p><p><mi>A</mi></p><p>int</p><p><mi>l</mi><mi>f</mi></p><p></p><p></p><p></p><p></p>) and the average number of inter-subunit hydrogen bonds during the simulations (<p></p><p></p><p></p><p><mi>N</mi></p><p><mi>H</mi><mo>−</mo><mi>b</mi></p><p><mi>e</mi><mi>q</mi></p><p></p><p></p><p></p><p></p>) are also indicated.<p></p><p>Overall effective interaction energy (</p><p></p><p></p><p></p><p><mi>W</mi></p><p>int</p><p><mi>e</mi><mi>q</mi></p><p></p><p></p><p></p><p></p>) and the contribution of non-bonded van der Waals (<p></p><p></p><p></p><p><mi>E</mi></p><p><mi>v</mi><mi>d</mi><mi>W</mi></p><p><mi>e</mi><mi>q</mi></p><p></p><p></p><p></p><p></p>) and Coulomb (<p></p><p></p><p></p><p><mi>E</mi></p><p><mi>C</mi><mi>o</mi><mi>u</mi></p><p><mi>e</mi><mi>q</mi></p><p></p><p></p><p></p><p></p>) interaction energy terms and of the solvation free energy change upon dimerization (<p></p><p></p><p><mi>Δ</mi></p><p><mi>G</mi></p><p><mi>s</mi><mi>o</mi><mi>l</mi><mi>v</mi></p><p><mi>e</mi><mi>q</mi></p><p></p><p></p><p></p><p></p>) averaged over all frames of the equilibrated phase of the productive MD simulations.<p></p

Additional file 2: Figure S2. of Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells

The representative photos of p-mTOR, p-S6, LDH-A and Gls immunostainings in HT-1080 xenograft tumours. The expressions of p-mTOR, p-S6, LDH-A and Gls were studied in control and Rapamune treated xenograft tumours (DAB-brown staining; magnification 400X) (PDF 209 kb)