Structural and Biochemical Characterization of Apicomplexan Inorganic Pyrophosphatases

Inorganic pyrophosphatases (PPase) participate in energy cycling and they are essential for growth and survival of organisms. Here we report extensive structural and functional characterization of soluble PPases from the human parasites Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase). Our results show that PfPPase is a cytosolic enzyme whose gene expression is upregulated during parasite asexual stages. Cambialistic PfPPase actively hydrolyzes linear short chain polyphosphates like PPi, polyP3 and ATP in the presence of Zn2+. A remarkable new feature of PfPPase is the low complexity asparagine-rich N-terminal region that mediates its dimerization. Deletion of N-region has an unexpected and substantial effect on the stability of PfPPase domain, resulting in aggregation and significant loss of enzyme activity. Significantly, the crystal structures of PfPPase and TgPPase reveal unusual and unprecedented dimeric organizations and provide new fundamental insights into the variety of oligomeric assemblies possible in eukaryotic inorganic PPases.

levels of PP i , accounting for the essential nature of this enzyme. In C. elegans, a null mutant of PPase was developmentally arrested at the larval stage with defects in intestinal morphology 11 . Mutant PPases were also found to be associated with cell cycle arrest and cell death in fermenting yeast 12 . Increased expression and activity of cytosolic PPase has been linked with aging in rat and mouse 13 . In humans, over-expression of cytosolic PPase is associated with many types of cancer such as those of breast and lung, ovarian, and hepato-carcinoma 14-17 . Due to their essential roles in metabolism, PPases have been studied as potential drug targets with a focus on pathogenic organisms. For example, a novel series of anti-PPase small molecules were shown to target drug resistant strains of Staphylococcus aureus 18 . In another study, selective inhibition of short-chain polyP activity of VSP1 by small molecule inhibitors provided protection against T. brucei infection in a mouse model 19 . Furthermore, a distinct allosteric site has been exploited to target M. tuberculosis PPases 20 . These studies show that PPases can be targeted at multiple structural levels, and offer hope of obtaining inhibitors by utilizing distinct structural and functional properties of PPases. Recently, our group elucidated the atomic structure of T. brucei VSP1 and highlighted several of its distinct features that may have implications for inhibitor design 21 . The soluble PPase from Toxoplasma gondii (TgPPase) has also been studied biochemically in the past 22 .
In present study, we focused on a previously uncharacterized P. falciparum soluble inorganic pyrophosphatase (PF3D7_0316300.1) referred to as PfPPase from hereon. Comparative sequence and domain analysis data suggested that PfPPase consists of 380 amino acids and differs markedly from homologous enzymes in its N-terminal region that is extended by ~76 amino acid rich in asparagines (~30% of the region), a feature often associated with low-complexity regions in P. falciparum 23 . In contrast, the N-terminal region of TgPPase (residue 1-78) is rich in glycine and serine residues. We report structural and biochemical characterization of these two apicomplexan PPases. We also present insights into the dimerization modes of eukaryotic family I soluble PPases.

Results and Discussion
Characterization of P. falciparum PPase expression levels. Reverse transcription PCR-based (RT-PCR) expression profile of the gene corresponding to PfPPase (PF3D7_0316300.1) suggested that PfPPase was transcribed during all asexual stages of P. falciparum; its expression increased relative to ring stage, during late trophozoite (LT)/early schizont (ES) and reduced again when schizonts mature (Fig. 1a). This gene expression profile was complimented by western blotting data that showed protein expression during all three stages but with higher expression during the trophozoite stage (Fig. 1b). Our quantitative PCR (qPCR) analyses using threshold C T supported our semi-quantitative PCR and western blotting profiles (Fig. 1c). The qPCR data suggested that expression of PfPPase increased ~4 fold as parasite rings transformed into ET (Fig. 1c). Interestingly, maturation of ET into LT/ES was accompanied by further increase in expression, which was ~10 fold higher relative to in rings (Fig. 1c). Eventually, the expression was restored to near basal levels in mature schizonts (Fig. 1c). Therefore, our results indicated that PfPPase levels were differentially regulated during asexual stages of P. falciparum, and stimulation of PfPPase expression could be attributed to anabolic nature of LT/ES stages that display high rates of protein synthesis and DNA replication.
The cell extracts derived from different P. falciparum asexual stages actively hydrolyzed PP i in presence of 1 mM Mg 2+ (Fig. 1d). In addition, this activity was inhibited completely by 1 mM NaF, a known inhibitor of PPase activity (Fig. 1d). These results further supported our gene/protein expression data that indicated presence of a functional PPase in P. falciparum. For functional and structural analysis of PfPPase, the enzyme was over-expressed and purified from E. coli. SDS-PAGE analysis of PfPPase confirmed its theoretical molecular weight (M wt ) ~45 kDa (Fig. 1e). However, the trace on gel filtration chromatogram showed that PfPPase existed predominantly as a dimer in solution with a minor tetrameric peak (Fig. 1f).
PfPPase is a cytosolic enzyme. To investigate localization of PfPPase in asexual stages of malaria parasites, we performed indirect immunoflorescence assays (IFA) using antibodies against purified recombinant PfPPase protein and a previously described protocol 24 . As evidenced by co-localization of Pf-Ed-VRS (valine-tRNA synthetase, a known cytosolic maker), the endogenous localization of the PfPPase enzyme was cytosolic (Fig. 2a). In addition to the cytosol, IFA studies with organelle markers (acyl-carrier protein-green fluorescent protein and mitotracker) revealed that PfPPase was not localized either in the apicoplast or the mitochondria ( Fig. 2b and c) in asexual parasite stages. Western blot analysis of cytosolic and membrane fractions of trophozoites further confirmed cytosolic localization of PfPPase (Fig. 2d). Consistent with this, the total activity of the enzyme was predominantly in the soluble fraction obtained from parasites (Fig. 2e).
Kinetic analysis of PfPPase and substrate binding. We examined PfPPase activity using a colorimetric assay for phosphate estimation 25 . PfPPase was found to be capable of utilizing PP i , polyP 3 and ATP as substrates (Fig. 3a,i-iii). PP i hydrolysis by PfPPase showed an absolute requirement for divalent cations and had a turnover number (k cat ) of 266 s −1 at 37 °C and optimum pH of 7.2 in presence of 3 mM Mg 2+ (Fig. 3a,i). Other divalent cations such as Co 2+ , Zn 2+ and Mn 2+ stimulated PP i hydrolysis but with lower efficiency ( Table 1). The relative PfPPase PP i activity conferred by divalent metal ions fell in the order Mg 2+ > Co 2+ ~ Zn 2+ > Mn 2+ . However, Zn 2+ was the preferred co-factor for hydrolysis of polyP 3 and ATP (Table 2). PfPPase displayed a K m of ~64 µM for ATP and it is noteworthy that the intracellular concentration of ATP in P. falciparum is in milimolar range, which thus suggests physiological significance of the above K m 26 . Overall; these data highlight the cambialistic properties of PfPPase. However, it is clear from k cat /K m values that the catalytic efficiency of PP i hydrolysis is higher than for other substrates (Tables 1 and 2). Surprisingly, Mg 2+ failed to stimulate hydrolysis of both polyP 3 and ATP. In a previous study Zyryanov et. al., had shown that the rate determining step in ATP hydrolysis was breakage of the P-O bond by PPases from S. cerevisae, E. coli, S. mutans and rat liver 27 . They further showed that higher efficiency of transition metal ions compared with Mg 2+ emanated from stronger binding of the terminal phosphate of ATP or polyP 3 in presence of the transition metal, which allowed more favorable position for catalysis 27 . We probed this idea using protein thermal shift (PTS) assays that provide an assessment of the stability of protein and protein-ligand complexes based on melting temperature (T m ) 28 . PTS analysis revealed substantial difference in AMPPNP (a substrate analog of ATP) binding in presence of transition metals and Mg 2+ ions. As shown in Table 3 and Fig. 3b(i-iv), the T m shifts suggested a higher affinity for Zn 2+ -AMPPNP (ΔT m = 10.3 °C) than Mg 2+ -AMPPNP (ΔT m = 0.8 °C). Moreover, the order of T m shift with AMPPPNP was same as the order of catalytic efficiency of ATP i.e. Zn 2+ > Co 2+ > Mn 2+ > Mg 2+ . These results thus validate and reaffirm the link between catalytic efficiency and strong binding of ATP or polyP 3 in presence of transition metals.

Crystal structures of PfPPase and TgPPase.
To obtain a comprehensive structural description of PfPPase and TgPPase we determined their crystal structures. We were successful in obtaining crystals of seleno-methionine (Se-Met) labeled PfPPase and hence used single wavelength anomalous (SAD) technique to obtain the crystal structure of PfPPase. The final structure was refined to highest resolution of 3.2 Å with R work / R free of 0.22/0.27 ( Table 4). Crystals of PfPPase belonged to space group C2 with five molecules in the asymmetric unit and Mathew's coefficient of V m ~ 3.0. Overall, the PfPPase crystal structure showed simple domain architecture, typified by five stranded β-barrel β 4 and β 7 -β 10 (Fig. 4a). This β barrel is flanked by helices α 3 (residue; 264-280) and α 5 (residue; 293-316). There are two 3 10 helical turns η 1 and η 2 at residues 144-146 and 258-251. The first 36 residues of PfPPase are missing in the current crystal structure and thus the structure extends from residues 37 to 380. The N-terminal region of PfPPase extends from 36-76 residues. This region is composed of a stretch that lacks secondary structure (residues 36-54), a small highly hydrophobic helical region (α 1 , residues 54-60) that is then followed by a strand (β 1 , residues 70-76) that leads into the PfPPase domain ( Fig. 4b-c). Residues 324-352 are highly disordered showing no clear density in the crystal structure. We next determined the crystal structure of TgPPase using molecular replacement method using ScPPase (PDB ID = 1WGJ) as a template. The TgPPase crystal structure was refined to 2.35 Å and showed the same structural overall fold as PfPPase. However, TgPPase lacked both N-and C-terminal regions and the structure contains residues 74-308 (Table 4 and Supplementary Figure 1a), most likely due to proteolysis of N-terminal region during crystallization. Structural homology searches using DALI server with PfPPase and TgPPase indicated that both structures show high similarity to PPase domain of T. brucei VSP1 (TbVSP1 PDB: 5C5V; Z = 30) and ScPPase, PDB: 1WGJ; Z = 18). Subsequent structural comparisons revealed that architectural differences between PfPPase, TgPPase, ScPPase and TbVSP1 existed mostly in surface areas such as the connections or loops between helices and strands (Fig. 4d). An interesting and a key distinct feature of PfPPase is its N region (residues 36-76) that stretches away from the structural core and is absent in both T. brucei and S. cerevisae PPases ( Fig. 4c-d). Interestingly, our data showed that deletion of the N-terminal region (residues 1-76) from PfPPase (ΔN-PfPPase) resulted in PPase aggregation and loss of enzyme activity ( Supplementary Fig. 2a). Furthermore, the thermal stability of the ΔN-PfPPase could not be derived as the fluorescent probe (SYPRO orange) was already bound to the protein at room temperature, an indication that protein was likely already unstable (Supplementary Figure 2b).

Active sites in PfPPase and TgPPase. Structural comparisons of PfPPase and TgPPase with ScPPase/
TbVSP1 revealed that residues responsible for binding of PP i and of Mg 2+ were located on the top of β-barrel and active site residues within were highly conserved (Supplementary Figure 3a-d and Fig. 4c). Crystals of PfPPase were grown in high concentration of PP i and in presence of Mg 2+ though we were unable to assign Mg 2+ . PfPPase active site showed electron density for only one P i molecule in two (B and C) out of five subunits (A-E) in the asymmetric unit (Supplementary Figure 3e). In structural comparisons with TbVSP1 and ScPPase, this P i molecule in PfPPase closely corresponded to the location of P i molecule of the bound PP i that is not directly attacked (P1) (Supplementary Figure 3a). This observation suggested that the directly attacked phosphate group (P2) of the PP i was first to dissociate from the active site of PfPPase. In contrast to PfPPase, the active site of TgPPase contained two bound Mg 2+ ions (Fig. 5b). One Mg 2+ was bound at M1 site coordinated by Asp190, Asp195 and Asp227 (Supplementary Fig. 2b). The Mg 2+ was bound to protein in M2 site predominantly through water molecules and Asp195 ( Supplementary Fig. 2b). However, no electron density for either P i or PP i was observed in the TgPPase active site despite addition of PP i during crystallization. Therefore, it is feasible that the observed conformation in the active site represents a state where both P i s have already dissociated.
Based on cues from crystal structures of these apicomplexan PPases, we tested the functional importance of selected active site residues by measuring enzyme kinetics of wild type and mutant PfPPase. We generated point mutations of three Asp residues (Asp198, Asp203 and Asp235) to Asn residues along with conversions of Lys136 and Arg158 to Arg and Lys respectively. As noted from the kinetic parameters calculated at 1.4 nM PfPPase enzyme concentration, the D235N and D198N perturbed activities modestly (~5 fold and ~6 fold reduction in    Table 3. Melting temperature (T m ) of PfPPase with divalent cations and AMPPNP.
k cat , respectively) and therefore may not play major catalytic roles (Table 5). In contrast, enzymatic activity of D203N was ~600 fold lower at aforementioned PfPPase enzyme concentration, suggesting its major catalytic role in PP i hydrolysis (Table 5). These results are in agreement with previous findings on ScPPase 29,30 . Comparison of PfPPase with TgPPase suggested that Asp203 was a structural counterpart of Asp195 in TgPPase where TgPPase Asp195 simultaneously co-ordinates M1 and M2 (Supplementary Figure 2b). It is known that two Mg 2+ ions in M1 and M2 site bridge a water molecule between them, generating the reaction nucleophile 29 . Therefore, D203N mutation might have impaired Mg 2+ binding to PfPPase that thus perturbed the nucleophile generation and hence the hydrolysis rate. In contrast to Asp mutants, the K136R variant of PfPPase showed a drastic 40-fold increase in K m indicating an overall compromise in affinity for PP i , along with 10 fold reduction in catalytic turnover of PP i hydrolysis (Table 5). By contrast, R158K was less marked and only modestly perturbed the k cat and K m ( Table 5). These results suggest possible loss of favorable interactions between mutant residues and PP i , which thus impair the catalytic rate by unfavorable positioning of electrophilic P i in the P2 site with respect to the catalytic water.
Thus, mutational probation of active site of PfPPase confirms the functional relevance of the active site residues. Given the fact that PfPPase and yeast PPase active sites are identical the published catalysis model is applicable to PfPPase as well 29-31 . Dimeric crystal structures of PfPPase and TgPPase. In the asymmetric unit of PfPPase crystals, four chains formed two non-crystallographic dimers, and dimerization of the fifth monomer was mediated via the crystallographic 2-fold axis. This dimeric crystal structure of PfPPase was consistent with our gel filtration and BN-PAGE data, though gel filtration also suggested a small fraction of tetrameric form that was not observed in the crystal structure. The dimensions of the observed PfPPase dimer are ~98 Å × 48 Å × 58 Å and it is arranged such that the α 5 helices in the two monomers lie anti-parallel to each other (Fig. 5a). The area for buried surfaces at dimer interfaces is ~ 2650 Å 2 per subunit. There are two distinct dimer interfaces that lie opposite to each other in the assembly -one face has the strap-like PfPPase N-region (residues 36-69), whereas the other has α 3 helix (Fig. 5a). In the N-region, α 1 helices of each subunit together form a cluster of hydrophobic residues (Fig. 5b). Interfacial interactions that exists at this hydrophobic patch are as follows; (1) aliphatic-aliphatic hydrophobic interactions between Ile55 and Ile59 (ii) aliphatic-aromatic interactions between Ile55 and Tyr160, and (iii) π-π stacking force between Phe50 (Fig. 5b). This hydrophic patch is stabilized by a network of hydrogen bonds formed between asparagines [a cluster of residues rich in asparagines (Asn49, Asn52-54, Asn58) and Asn62] of N-region with PPase domain (Fig. 5b and c). All interfacial asparagines have self evident electron densities and all interfacial residues are highly conserved and but limited to the Plasmodium species only (Fig. 5d and e). This N-region  Table 4. Data collection and Refinement statistics.
contributes ~1800 Å 2 to average buried surface area at interface, and this data supports its consideration as a physiologically relevant interface. The second interface engages α 3 helix of the PPase domain from each subunit (Fig. 5e). The side chain interactions that stabilize this interface are hydrogen bonds between Ser266 and Glu270 and NH…π between Arg274 and His263 (Fig. 5f). Like PfPPase, TgPPase also formed dimers in solution and in the crystal (Supplementary Figure 1b). Two TgPPase subunits buried 1780 Å 2 (50%) of total ASA and are held together mainly by a network hydrogen bonds and Van der Waal forces. Some notable interactions include Secondary structure elements (yellow arrow:β-sheet; blue rectangular boxes; α-helices); yellow triangles (pyrophosphate) and pink circles (metal binding) indicate active site residues; (d) Structural superposition of PfPPase onto TbVSP1, ScPPase, TgPPase reveals conservation of core structure, with differences in peripheral parts of the structure. N-terminal extension of PfPPase is indicated by arrow.

Comparison of Eukaryotic Family I PPases reveal diversity in dimerization modes. Previously,
the oligomeric assembly of eukaryotic family I PPases was reported to be mostly dimeric before our analysis of TbVSP1 showed a tetrameric arrangement 21 (dimer of dimers). Interestingly, the thus far studied dimeric PPases are different from each other in their monomer-monomer contacts as revealed by their buried surface areas; amongst the known set from PPase structures in PDB the PfPPase forms the tightest dimer as judged by PISA analysis (Fig. 6a). This prompted us to analyze the underlying structural features responsible for the disparity atomic embraces displayed by dimeric PPases. In the crystals of TbVSP1, each monomer packs against two other   monomers having two different interfaces 21 . The small dimer DI (830 Å 2 ) forms via loops between strands β 6 -β 7 and β 4 -β 5 21 . The larger dimer-DII (1407 Å 2 ) forms mainly via extensive contacts between a "long loop" that connects β 8 -β 9 and residues from β 1 and β 7 of the PPase domain 21 (Fig. 6b and c). By contrast, crystal structure of dimeric ScPPase (930 Å 2 ) reveals that its monomers are held by their C-terminal extensions, which are important for stability of the dimer 21,29 (Fig. 6d). As revealed in previous sections, for PfPPase its N-region and α 5 helix are two predominant structural elements that assemble the PfPPase dimer (Fig. 6e). On the other hand, TgPPase monomers are joined by α 3 only (Fig. 6f). From aforementioned structural comparisons, it is clear that Pf and TgPPase dimerize differently. Further, there is strong variance in how TbVSP1 (α 7 ) and ScPPase (α 4 ) associate (Fig. 6b,c and d). In order to visualize these different modes of PPase dimerizations, we superimposed the dimeric structures of PPases in a way that the position for one monomer is fixed (Fig. 7a-d). PfPPase was used as reference dimer and rotational differences suggested that position of second monomer of TbVSP1 (DI) and ScPPase varied considerably, while the dimerization modes of PfPPase and TgPPase are more similar (Fig. 7c). Strikingly, TbVSP1 (DII) showed different spatial position of the second monomer (Fig. 7d). This could be attributed to the observation that the oligomerization face of DII-TbVSP1 is on the opposite face of PPase domain with respect to the active site, as opposed to in other PPases, which have it on the same face as the active site (Fig. 8a). The differences in subunit orientation in Tb, Tg, Sc and Pf PPase dimers are likely to originate from the diversity in sequence of amino acids contributing to the dimerization interfaces (Fig. 8b). It is evident from the sequence alignments that there are very few overlapping (conserved) interfacial residues (Fig. 8b). Dissimilar modes of association could also be attributed to specific secondary structure elements that are involved in dimerization (Fig. 8b). For example, a long loop (residues 198-223) is present in TbVSP1 and is conserved across kinetoplastida, however, TbVSP1 lacks the C-terminal extension of fungal PPases 21 . Similarly, a strap-like N-region extension seems limited to Plasmodia. Although a C-terminal extension similar to ScPPase is also present in the primary sequence of PfPPase and TgPPase, it is not involved in dimer formation of either of them, as revealed by their crystal structures and gel filtration analysis (Fig. 8b and Supplementary Figure 4). Therefore, comparative

Conclusions
Although the canonical domain architectures of eukaryotic soluble PPases seems well conserved, there is increasing structural evidence for divergence in their oligomeric assemblies via gain, loss or extension of N/C terminal regions in their sequences. Here we have demonstrated that the N-region of PfPPase is indispensable for enzyme stability and oligomeric integrity. We have compared and contrasted crystal structures of apicomplexan, yeast and kinetoplastid PPases. We also reveal a general heterogeneity in the dimerization modes of these eukaryotic PPases. This study thus provides a detailed architectural glimpse of apicomplexan PPases, and we hope that it will be useful in supporting future studies on phosphate metabolism pathways in these parasites.

Methods
Production of PfPPase and TgPPases. The ORFs of full length PfPPase (residues 1-380) were cloned in pETM41 using NcoI and KpnI restriction sites. Transformed E. coli BL21-CodonPlus was grown in LB medium containing 50 μg/ml kanamycin to an OD 600 of 0.6-0.8 at 37 °C. Expression of the recombinant proteins was induced by the addition of 0.5 mM isopropyl β-D-galactoside, and incubation was continued for a further 20 h at 18 °C. The recombinant PfPPase bears a MBP-6X histidine tag. Briefly, bacterial cells were lysed by sonication in a buffer (100 mM HEPES-Na pH 7.2, 500 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol) containing protease inhibitor. Affinity purification was performed on amylose resin (NEB) and Ni-NTA (His-Trap FF, GE healthcare) using an AKTA FPLC system. Both tags were cleaved with TEV protease followed by dialysis in low salt buffer (30 mM HEPES pH 7.4, 30 mM NaCl, 1 mM DTT). Protein was subsequently applied to Q-Sepharose (GE healthcare) column for further purification and removal of TEV protease. Finally, pure fractions were pooled and concentrated to 10 mg ml −1 with 10 kDa cutoff centrifugal devices (Millipore) followed by Gel Permeation Chromatography (GPC) on S-200-16/60 column (GE-healthcare) in a buffer containing 30 mM HEPES-Na pH 7.2, 100 mM NaCl and 1 mM DTT. TgPPase was cloned in pETM11 and purified using Ni-NTA chromatography followed by Q-sepharose ion exchange chromatography and GPC.
Real-Time PCR. Total RNA was extracted from 3D7 P. falciparum cells using intra-erythrocytic parasite stages and by trizol method. For time-course studies, P. falciparum parasites were taken at 8-12 h, 24-28 h, Scientific RepoRts | 7: 5255 | DOI:10.1038/s41598-017-05234-y 34-42 h and 42-48 h post synchronization representing rings, early trophozoite, late trophozoite/early schizont and mature schizonts -as confirmed by microscopy. Total 1.5 µg of total RNA was amplified in 42 µl reaction volume using oligo dT primers and Superscript III reverse transcription kit (InVitrogen) following which the reaction mix was diluted ten times prior to real-time amplification. To study temporal expression of predicted PfPPase gene, real time PCR was performed on ABI step one plus (Applied Biosystems) using Quantitect SYBER Green I mix (Qiagen, Hamburg, Germany). Threshold cycle (C t ) values were determined using ABI prism software and 2 −ΔΔCt was used to calculate relative expression values. PfSerRS (Seryl-tRNA synthetase) gene was the internal reference control and ΔC t value of the ring stage was used as the calibrator. Primers used for amplification were ~200 bp in size and were first optimized to give maximum amplification. The amplification factor for primers of PfPPase and PfSerRS were 1.96 and 1.98 respectively.

Measurement of kinetic activity of PfPPase.
PfPPase activity was measured based on methods described previously 25 . PfPPase was added to the reaction mixture-carrying varying concentrations of substrates (PP i , polyP 3 and ATP) in reaction buffer suitable for optimum pH with 100 mM NaCl at 37 °C at for 5 min. The optimum pHs for PP i , polyP 3 and ATP hydrolysis were 7.2, 7.0 and 7.2 respectively. To stop enzymatic reactions, one volume of malachite green reagent was mixed with four volumes of enzymatic reaction to be analyzed. The mixture was Figure 8. Strategies of diversification in oligomerization modes. (a) Crystal structures of PPase monomers colored in rainbow color (monomer's spectrum runs through blue/cyan to red i. e. from N-terminus to C-terminus) showing oligomerization faces (orange bracket). Interfacial residues are mapped as spheres onto the structure at the equivalent C α residues and orientation of active site is indicated by purple arrow. (b) Sequence alignment of eukaryotic PPases where crystal structures are known. The interfacial residues (i) colored according to the species are mapped on to the sequence from structural data; specific and unique structural elements are boxed. incubated for 3 min and absorbance at 623 nm was measured with a spectromax UV/VIS spectrophotometer (BIO RAD). To measure activity in total cell lysate, isolated malaria parasite trophozoites were washed with buffer containing 30 mM HEPES pH 7.2, 116 mM NaCl, 5 mM KCl, 5 mM glucose (to prevent premature cell lysis and release of proteases) and resuspended in the same buffer. The cells were broken by sonication (20% amplitude, five, 2 seconds pulses). The activity was also measured from sub-cellular fractions obtained via lysis with 1% Triton-X-100 as described previously 32 . Each data point was produced from individual experiments that were performed in triplicates. Absorbance values were read twice using spectrophotometer to ensure integrity of the data.
Protein Thermal Shift Assays. These were performed with 2.5 μM PfPPase in 30 mM HEPES-Na, pH 7.2, 100 mM NaCl and a 1× dilution of SYPRO orange dye (Invitrogen). The dye was excited at 490 nm and emission light was recorded at 575 nm while the temperature was increased in increments of 1 °C per minute from 20-98 °C. Control assays were carried out in the absence of protein or dye to ensure that no fluorescence signal was recorded. Thermal shift experiments of PfPPase complexes were performed using analogs Imidodiphosphate (PNP) (Sigma Aldrich) and AMPPNP (Sigma Aldrich). These are chemical mimics of PP i and ATP respectively. Both AMPPNP was used at 5 mM concentration and mixed with 1 mM of each Mg 2+ , Co 2+ , Zn 2+ and Mg 2+ .
Immunolocalization and western blotting. Immunofluorescence assays were performed using protocols described previously 24 . Purified primary rabbit anti-PfPPase antibody at 1:200 dilution was used. Secondary antibodies used were Alexa flor 545 (Invitrogen) and Alexa flor 595 (Invitrogen). Mitotracker Red CH 2 X ROS was used to stain the parasite mitochondria. For western blotting experiments, proteins were separated on SDS-PAGE gel and analysis was performed using anti-PfPPase (1:500). Rabbit Anti-PfNAPL (1:2000) antibodies were used as internal control 33 .

Crystallization of PfPPase and TgPPases.
A single peak corresponding to dimer of PfPPase was collected from GPC. This protein solution contained 3 mM MgCl 2 and 1 mM PP i (Sigma Aldrich) and was used for co-crystallization (10 mg ml −1 ). Crystallization conditions were initially sought by vapor diffusion method at 293 K using commercially available crystallization screens. Crystals of seleno-methionine substituted PfPPase were obtained in buffer condition 8% Tacsimate pH 8.0 and 20% PEG3350. Multiple single crystals were obtained for the protein in drops ~4 days. PfPPase crystals were cryo-protected with 18% ethylene glycol in mother liquor prior to mounting. TgPPase (concentrated to 10 mg ml −1 ) crystals were obtained within six to eight days in MORPHEUS crystallization screen (Molecular Dimensions). The well condition corresponded to 10% PEG4000, 20% glycerol, 0.03 M glycols and 0.1 M HEPES/MOPS pH7.5.
Structure determinations and refinements. Selenomethionine (Se-Met)-labeled PfPPase crystals were protected by a cryoprotectant containing 8% Tacsimate pH 8.0 and 20% PEG3350, and the data were collected at BM14, ESRF, Grenoble at the peak wavelength of 0.9762 Å at 100 K. The dataset was indexed, integrated and scaled using HKL2000 34 . The Se-Met crystals of PfPPase belonged to monoclinic space group C2 with cell dimensions of a = 253.19, b = 85.23, c = 108.45 Å with five molecules in the asymmetric unit (ASU). 22 Selenium were identified in the ASU using HySS 35 and subsequent phasing was done using Autosol 36 . Autosol derived phases were used to build a partial model with AutoBuild 37 . The complete model was built using Coot 38 and the model was refined with phenix.refine 39 .
Native data were collected for TgPPase at BM14, ESRF, Grenoble. TgPPase crystal structure was determined by molecular replacement method with the program PHASER 40 within the PHENIX suite, using yeast soluble PPase as the search model (PDB code = 1WGJ), with 44% sequence identity from residue 107 to 304 of the target. The results from molecular replacement for TgPPase showed a translation function Z-score of 15 that strongly suggested a correct solution. The atomic positions obtained from molecular replacement and the resulting electron density maps were used to build (AutoBuild and Coot) the TgPPase structures and initiate crystallographic refinement (phenix.refine). The coordinates and structure factors for PfPPase and TgPPase have been deposited in the PDB under accession code 5WRU and 5WRT respectively. Structure figures were generated using CHIMERA 41 & PyMOL (Schrodinger).