Fig 6. Western Blot analysis demonstrates the ability of His₆-tagged <i>Pf</i>MDH-V190W mutants to incorporate into pre-formed native Strep-tagged oligomeric assembly post-expression.

<p>Both proteins were recombinantly expressed and the lysate were mixed and sequentially purified via Ni-NTA and Strep-tactin chromatography. (Left) Mixed lysate and wild-type <i>Pf</i>MDH were first purified via Strep-Tactin (IBA Lifesciences) chromatography and subsequently analyzed by Western Blot using α-strep antibodies (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#sec009" target="_blank">Materials and methods</a>), confirming the presence of Strep-tagged <i>Pf</i>MDH-WT in both samples. (Right) Strep-purified samples were further purified via Ni-NTA chromatography. Western Blot with α-His antibodies showed no signal for the wild type sample (as expected) and confirmed the presence of His₆-tagged V190W mutant in co-purified sample.</p

Data collection and refinement statistics of <i>Pf</i>MDH.

<p>Data collection and refinement statistics of <i>Pf</i>MDH.</p

Summary of the biophysical parameters of <i>Pf</i>MDH mutants, as assessed by Static Light Scattering assay (SLS), Microscale Thermophoresis assay (MST) and Thermal Shift Assay (TSA).

<p>Summary of the biophysical parameters of <i>Pf</i>MDH mutants, as assessed by Static Light Scattering assay (SLS), Microscale Thermophoresis assay (MST) and Thermal Shift Assay (TSA).</p

Primers used in this study.

<p>Primers used in this study.</p

Sequence conservation of <i>Pf</i>MDH across homologs and surface analysis.

<p>Sequence conservation of <i>Pf</i>MDH across homologs and surface analysis.</p

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of <i>Plasmodium falciparum in vitro</i> - Fig 1

<p>Fig 1 (a) shows the secondary structure of wild type <i>Pf</i>MDH as well as substrate and cofactor binding sites. Like other malate dehydrogenases, <i>Pf</i>MDH consists of 9 <i>alpha</i>-helixes and 11 <i>beta</i>-sheets. First 6 <i>beta</i>-sheets form parallel structure (Rossman fold) and belong to the cofactor-binding N-terminal domain. The NADH and pyruvate molecules were modeled using superposition with homologous MDH structure (<u><b>4PLC</b></u>, rmds 1 Å) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref026" target="_blank">26</a>]. (b) <i>Pf</i>MDH is a globular homo-tetramer, the subunits are labeled A, B, C and D. (c) Structural superposition of <i>Pf</i>MDH (green) with predicted ancestral apicomplexian malate dehydrogenase (53% sequence identity, 1 Å rmsd on C-<i>alphas</i>, <u><b>4PLC</b></u>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref026" target="_blank">26</a>] shown in magenta. (d) Structural superposition of <i>Pf</i>MDH (green) with MDH from <i>Cryptosporidium parvum</i> (43% sequence identity, 1.3 Å rmsd on C-<i>alphas</i>, <u><b>2HJR</b></u>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref027" target="_blank">27</a>] used for molecular replacement (cyan). Structures were superimposed using GESAMT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref048" target="_blank">48</a>] package from CCP4 suite [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref044" target="_blank">44</a>] and visualized using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref049" target="_blank">49</a>].</p

Fig 5 shows specific activity (both forward and reverse reactions) of the wild type <i>Pf</i>MDH, E18Q, V190W, E18W mutants, and co-purified WT-mutant chimeric complex.

<p>Specific activity is shown in Units per mg of enzyme (U mg^-1). U = μmol of NADH oxidized or (NAD⁺) reduced per minute. (a & b) Interference with the native oligomeric state of <i>Pf</i>MDH affected substrate kinetics as well as significantly changed specific activity of the mutant species. (b) E18Q mutant showed increased specific activity for reverse reaction as well as significant substrate inhibition at substrate concentrations above 2.5 mM. (c) At 1.6 mM malate (reported intracellular substrate concentration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref035" target="_blank">35</a>]) dimeric <i>Pf</i>MDH mutants had significantly reduced specific activity towards malate oxidation, while E18Q mutant activity was not significantly changed compared to WT. No activity could be detected for co-purified <i>Pf</i>MDH-WT/V190W chimeric assembly. (d) At sub-millimolar substrate concentration (0.625 mM oxaloacetate) E18Q mutant showed significantly increased (10x) specific activity, while V190W mutant or co-purified WT/V190W chimera showed little or no measurable activity, respectively. Interestingly, E18W mutation disrupting AB interface resulted in slightly increased activity towards oxaloacetate reduction compared to the wild type enzyme.</p

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of <i>Plasmodium falciparum in vitro</i> - Fig 3

<p>Fig 3. (a-top) Undisrupted oligomeric interface of <i>Pf</i>MDH (AC). Subunit A is schematically shown as cartoon (yellow), surface of the adjacent subunit C is shown in cyan. (a-bottom) Interface mutation V190W located between α6 and β8 causes disruption of the A-C interface as confirmed by SLS experiments. Point V190W mutations were modeled in <i>Pf</i>MDH structure using Coot [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref046" target="_blank">46</a>] and visualized using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref049" target="_blank">49</a>]; mutated clashing residues are shown in red. (b-top) Native AB oligomeric interface of <i>Pf</i>MDH; Glutamate 18 pair in the core <i>α</i>1:<i>α</i>1 is shown in sticks. (b-bottom) Predicted steric clash caused by E18W mutation, causing an oligomeric disruption of AB interface and a model of the additional hydrogen bond pair (Gln18-Gln18) introduced between <i>α</i>1 helixes from adjacent subunits.</p

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of <i>Plasmodium falciparum in vitro</i> - Fig 2

<p>Figs 2 a-c show the interfaces formed between individual subunits of <i>Pf</i>MDH: AB (a), AC (b) and AD (c); residues involved in the oligomeric contact are shown in blue. Evolutionary conservation of the interface residues is shown in red (absolutely conserved), orange (strictly conserved) and green (slightly conserved). For more details on sequence conservation please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.t002" target="_blank">Table 2</a>. (d) Positions of the active sites of adjacent subunits A (yellow) and B (Magenta) are shown. Active sites from A and B subunits are mirror reflections of each other, well separated and distal to AC interface. (e) Structural superposition of <i>Pf</i>MDH AB subassembly (green) and dimeric malate dehydrogenases from <i>E</i>. <i>coli</i> MDH (29% sequence identity, 2.5 Å rmsd, <u><b>2PWZ</b></u>, primary citation unavailable) shown in gold. In order to highlight the active site positions, the NADH and pyruvate molecules were modeled into the active sites using superposition with homologous MDH structure (<u><b>4PLC</b></u>, rmds 1 Å) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref026" target="_blank">26</a>]. Structure superposition was performed using GESAMT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref048" target="_blank">48</a>] package from CCP4 suite [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref044" target="_blank">44</a>] and visualized using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195011#pone.0195011.ref049" target="_blank">49</a>].</p

Fig 4 shows examples of TSA melting curves of <i>Pf</i>MDH WT (dark bold lines), <i>Pf</i>MDH-V190W (dotted red lines), <i>Pf</i>MDH-E18W (dotted blue lines) and <i>Pf</i>MDH-E18Q (faint lines) in various buffer conditions: (a) PBS, (b) 400 mM NaCl, (c) 100 mM Na-Citrate pH 5.5 and (d) Assay Buffer (100 mM Na-Phosphate pH 7.4, 400 mM NaCl).

<p>Melting temperatures of each sample are displayed next to the respective curves. Analysis of these curves shows that <i>Pf</i>MDH is rather unstable in PBS (a) and requires optimized buffer conditions for further experiments. This effect is more pronounced for its mutant forms, where native oligomeric assembly has been disrupted (dotted lines). <i>Pf</i>MDH-E18Q mutant shows higher thermal stability, thus supporting the hypothesis that introduction of the additional hydrogen bond pair at the AB interface has had the desired stabilization effect. (b) 400 mM NaCl has significantly stabilized the wild type <i>Pf</i>MDH (ΔT_m = 10 K), dimeric V190W mutant (ΔT_m = 7 K) and tetrameric E18Q mutant (ΔT_m = 8.5 K), while having minor effect of the E18W dimeric mutant (ΔT_m = 2.5 K). (c) 100 mM Na-Citrate pH 5.5 significantly stabilized the wild type enzyme (ΔT_m = 17.5 K) and the E18Q mutant (ΔT_m = 15.5 K), while having lesser effect on V190W mutant (ΔT_m = 6 K) and negligible effect on E18W (ΔT_m = 0.5 K). (d) Selection of the Assay Buffer allowed further experiments to be performed for all four <i>Pf</i>MDH constructs used in this study in the same stabilizing buffer conditions (WT ΔT_m = 13 K, V190W ΔT_m = 7 K, E18W ΔT_m = 5 K, E18Q ΔT_m = 12.5 K). TSA assays were performed in 96-well PCR plates (VWR) using SFX96 Real-Time PCR reactor (BioRad). Melting curve (in terms of increased fluorescence, RFU) of each sample was plotted against the temperature gradient (293–363 K) using BioRad SFX96 software and the temperatures of the inflection points (T_m’s) were used as indicators of the thermal stability of each sample. ΔT_m’s reflect stabilization effect of each condition compared to the control experiments performed in PBS. For more details refer to Materials and Methods section.</p