Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance.

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei.

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg(2+) and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1.

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs.

Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.

Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.

Characterization of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) and its role in microneme secretion during erythrocyte invasion.

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu(356) to Thr(370)) of the JD in the presence of calcium (Ca(2+)). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.

Atomic resolution crystal structure of glutaredoxin 1 from Plasmodium falciparum and comparison with other glutaredoxins.

Glutaredoxins (Grxs) are redox proteins that use glutathione ((γ)Glu-Cys-Gly; GSH) as a cofactor. Plasmodium falciparum has one classic dithiol (CXXC) glutaredoxin (glutaredoxin 1; PfGrx1) and three monothiol (CXXS) Grx-like proteins (GLPs), which have five residue insertions prior to the active-site Cys. Here, the crystal structure of PfGrx1 has been determined by the sulfur single-wavelength anomalous diffraction (S-SAD) method utilizing intrinsic protein and solvent S atoms. Several residues were modelled with alternate conformations, and an alternate position was refined for the active-site Cys29 owing to radiation damage. The GSH-binding site is occupied by water polygons and buffer molecules. Structural comparison of PfGrx1 with other Grxs and Grx-like proteins revealed that the GSH-binding motifs (CXXC/CXXS, TVP, CDD, Lys26 and Gln/Arg63) are structurally conserved. Both the monothiol and dithiol Grxs possess three conserved water molecules; two of these were located in the GSH-binding site. PfGrx1 has several polar and charged amino-acid substitutions that provide structurally important additional hydrogen bonds and salt bridges missing in other Grxs.

ATP mimetics targeting prolyl-tRNA synthetases as a new avenue for antimalarial drug development.

The prolyl-tRNA synthetase (PRS) is an essential enzyme for protein translation and a validated target against malaria parasite. We describe five ATP mimetics (L95, L96, L97, L35, and L36) against PRS, exhibiting enhanced thermal stabilities in co-operativity with L-proline. L35 displays the highest thermal stability akin to halofuginone, an established inhibitor of Plasmodium falciparum PRS. Four compounds exhibit nanomolar inhibitory potency against PRS. L35 exhibits the highest potency of ∼1.6 nM against asexual-blood-stage (ABS) and ∼100-fold (effective concentration [EC50]) selectivity for the parasite. The macromolecular structures of PfPRS with L95 and L97 in complex with L-pro reveal their binding modes and catalytic site malleability. Arg401 of PfPRS oscillates between two rotameric configurations when in complex with L95, whereas it is locked in one of the configurations due to the larger size of L97. Harnessing such specific and selective chemical features holds significant promise for designing potential inhibitors and expediting drug development efforts.

Structural characterization of glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum, which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate L-Glutamate (L-Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (PfGluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of PfGluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the PfGluRS showed binding of the enzyme substrates L-Gluand ATP.. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the PfGluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs.

Utility of anion and cation combinations for phasing of protein structures.

We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination.

Structural delineation of histone post-translation modifications in histone-nucleosome assembly protein complex.

Nucleosome assembly proteins (Nap) are histone chaperones with vital roles in chromatin assembly and disassembly. Decoding of histone post-translational modifications by histone chaperones is central in regulation of gene expression. We probed binding interfaces in Nap-histone complexes using histone peptide interaction arrays and chemical shift perturbation techniques. Phosphorylation at H3T32 and H3T118 enhanced Nap-peptide interactions while post-translational modifications like H3K122, H4K59 and H2AK36 diminished it. Nap recognition regions in histones H2A, H2B, H3 and H4 lie in core regions of the histone octamer, and form a surface exposed contiguous patch. Nap binding sites on H2A-H2B and H3-H4 fall on opposite surfaces of histone octamer, and the latter can therefore accommodate two dimeric Nap molecules simultaneously. Interfacial residues between Nap and histone octamer overlap with route of wrapped DNA on histone octamer, implying non-concurrent residency on the octamer by either Nap or DNA. Using NMR, we mapped Nap residues that undergo chemical shift perturbations in presence of histone peptides. Taken together, we provide an architectural model of a Nap-octamer complex which likely forms during chromatin reorganisation.

A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases.

The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH2. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the Oδ2/Nδ2 of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRSD408E retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNAAsp with L-asp like wild type enzyme, while the PfDRSK372L was rendered enzymatically inactive. The correspondingly mutated PfNRSE395D was enzymatically inactive. The mutated PfNRSL360K had an altered specificity and esterified tRNAAsn with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.

Ligand-bound structures provide atomic snapshots for the catalytic mechanism of D-amino acid deacylase.

D-tyrosyl-tRNA(Tyr) deacylase (DTD) is an editing enzyme that removes D-amino acids from mischarged tRNAs. We describe an in-depth analysis of the malaria parasite Plasmodium falciparum DTD here. Our data provide structural insights into DTD complexes with adenosine and D-amino acids. Bound adenosine is proximal to the DTD catalysis site, and it represents the authentic terminal adenosine of charged tRNA. DTD-bound D-amino acids cluster at three different subsites within the overall active site pocket. These subsites, called transition, active, and exit subsites allow docking, re-orientation, chiral selection, catalysis, and exit of the free D-amino acid from DTD. Our studies reveal variable modes of D-amino acid recognition by DTDs, suggesting an inherent plasticity that can accommodate all D-amino acids. An in-depth analysis of native, ADP-bound, and D-amino acid-complexed DTD structures provide the first atomic snapshots of ligand recognition and subsequent catalysis by this enzyme family. We have mapped sites for the deacylation reaction and mark possible routes for entry and egress of all substrates and products. We have also performed structure-based inhibitor discovery and tested lead compounds against the malaria parasite P. falciparum using growth inhibition assays. Our studies provide a comprehensive structural basis for the catalytic mechanism of DTD enzymes and have implications for inhibition of this enzyme in P. falciparum as a route to inhibiting the parasite.

Structural analyses of the malaria parasite aminoacyl-tRNA synthetases provide new avenues for antimalarial drug discovery.

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl-tRNA synthetases (aaRSs) have been explored as anti-bacterial and anti-fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure-based knowledge of the most advanced three aaRSs-lysyl- (KRS), prolyl- (PRS), and phenylalanyl- (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.

Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines.

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.

Structure of D-tyrosyl-tRNATyr deacylase using home-source Cu Kalpha and moderate-quality iodide-SAD data: structural polymorphism and HEPES-bound enzyme states.

D-Tyrosyl-tRNA(Tyr) deacylase (DTD) is an editing enzyme that removes D-amino acids from mischarged tRNAs. The crystal structure of Plasmodium falciparum DTD (PfDTD) was determined using the iodide-SAD phasing method. Iodide-derivatized PfDTD crystals were obtained using the quick cryo-soaking procedure in which native crystals were soaked for a short period of 10-30 s in cryoprotectant solution containing 0.2-1 M NaI. Iodide-SAD data sets were collected to 3.3 and 2.74 A resolution from PfDTD crystals that belonged to two different space groups, P4(3) and P1, using an in-house X-ray copper-anode source. This is the first report to detail structure solution using low iodide anomalous signal, modest resolution and redundancy and average solvent content for SAD phasing of 984 and 1312 amino acids in the triclinic P1 and tetragonal P4(3) space groups, respectively. A total of 85% and 56% of the residues were automatically built into the iodide-phased electron-density maps using PHENIX AutoBuild. The structure of HEPES-bound PfDTD was subsequently determined by molecular replacement and refined to 2.83 A resolution. The crystals obtained from various batches of crystallization trials of PfDTD exhibited polymorphism in terms of belonging to different crystal forms and space groups. Even within a given crystal system the unit-cell parameters showed high non-isomorphism. These packing variations were exploited in order to conduct a systematic study of conformational changes in PfDTD. It is shown that the disposition of a ten-residue insertion loop affects packing within the PfDTD crystals and seems to determine the non-isomorphism in unit-cell parameters. By tracking the changes in PfDTD unit cells, it was possible to map conformational differences within PfDTD that may be of significance for enzyme activity.

Structural analysis of actinidin and a comparison of cadmium and sulfur anomalous signals from actinidin crystals measured using in-house copper- and chromium-anode X-ray sources.

The structure of the 24 kDa cysteine protease saru-actinidin from the fruit of Actinidia arguta Planch. (sarunashi) was determined by the cadmium/sulfur-SAD method with X-ray diffraction data collected using in-house Cu Kα and Cr Kα radiation. The anomalous scatterers included nine sulfurs and several cadmium ions from the crystallization solution. The high quality of the diffraction data, the use of chromium-anode X-ray radiation and the substantial anomalous signal allowed structure determination and automated model building despite both a low solvent content (<40%) and low data multiplicity. The amino-acid sequence of saru-actinidin was deduced from the cDNA and was modified based on experimental electron-density maps at 1.5 Šresolution. The active site of saru-actinidin is occupied by a cadmium ion and the active-site cysteine is found to be in an unmodified, cysteine sulfenic acid or cysteine sulfinic acid form. The cadmium sites, coordination geometries and polygonal water structures on the protein surface have also been extensively analyzed. An analysis and comparison of the sulfur/cadmium anomalous signals at the Cu Kα and Cr Kα wavelengths was carried out. It is proposed that the inclusion of cadmium salts in crystallization solutions coupled with chromium-anode radiation can provide a convenient route for structure determination.

Structure, localization and histone binding properties of nuclear-associated nucleosome assembly protein from Plasmodium falciparum.

BACKGROUND: Nucleosome assembly proteins (NAPs) are histone chaperones that are crucial for the shuttling and incorporation of histones into nucleosomes. NAPs participate in the assembly and disassembly of nucleosomes thus contributing to chromatin structure organization. The human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. METHODS: Three-dimensional crystal structure of PfNapS has been determined and analysed. Gene knockout and localization studies were also performed on PfNapS using transfection studies. Fluorescence spectroscopy was performed to identify histone-binding sites on PfNapS. Extensive sequence and structural comparisons were done with the crystal structures available for NAP/SET family of proteins. RESULTS: Crystal structure of PfNapS shares structural similarity with previous structures from NAP/SET family. Failed attempts to knock-out the gene for PfNapS from malaria parasite suggest essentiality in the parasite. GFP-fused PfNapS fusion protein targeting indicates cellular localization of PfNapS in the parasite nucleus. Fluorescence spectroscopy data suggest that PfNapS interacts with core histones (tetramer, octamer, H3, H4, H2A and H2B) at a different site from its interaction with linker histone H1. This analysis illustrates two regions on the PfNapS dimer as the possible sites for histone recognition. CONCLUSIONS: This work presents a thorough analysis of the structural, functional and regulatory attributes of PfNapS from P. falciparum with respect to previously studied histone chaperones.

Crystal structures of the two domains that constitute the Plasmodium vivax p43 protein.

Scaffold modules known as aminoacyl-tRNA synthetase (aaRS)-interacting multifunctional proteins (AIMPs), such as AIMP1/p43, AIMP2/p38 and AIMP3/p18, are important in driving the assembly of multi-aaRS (MARS) complexes in eukaryotes. Often, AIMPs contain an N-terminal glutathione S-transferase (GST)-like domain and a C-terminal OB-fold tRNA-binding domain. Recently, the apicomplexan-specific Plasmodium falciparum p43 protein (Pfp43) has been annotated as an AIMP and its tRNA binding, tRNA import and membrane association have been characterized. The crystal structures of both the N- and C-terminal domains of the Plasmodium vivax p43 protein (Pvp43), which is an ortholog of Pfp43, have been resolved. Analyses reveal the overall oligomeric structure of Pvp43 and highlight several notable features that show Pvp43 to be a soluble, cytosolic protein. The dimeric assembly of the N-terminal GST-like domain of Pvp43 differs significantly from canonical GST dimers, and it is tied to the C-terminal tRNA-binding domain via a linker region. This work therefore establishes a framework for dissecting the additional roles of p43 orthologs in eukaryotic multi-protein MARS complexes.