Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs, and confer conformational changes leading to modulation of functional parameters of their target proteins. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have annotated and characterized PF3D7_0818200 as 14-3-3 isoform I in Plasmodium falciparum 3D7, and its interaction with one of the key kinases of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Molecular dynamics simulation studies indicated that CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The 14-3-3I/CDPK1 interaction was validated in vitro with ELISA and SPR, which confirmed that the interaction is phosphorylation dependent, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: Mode I-type ARSHpSYPA and Mode II-type RLYHpSLPA, by simulation studies and ITC. This interaction was found to marginally enhance CDPK1 functional activity. Furthermore, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.
Cellular signal transduction pathways often involve Post-Translational Modifications (PTMs) of proteins which influence their overall spatial 3D-conformation, thereby affecting their stability, activity, and/or cellular localization . Reversible phosphorylation of serine, threonine or tyrosine residues has been the most extensively studied PTM . However, very often, phosphorylation is not solitary responsible for the modulation of protein function. Rather, phosphorylation of a protein mediates its interaction with downstream interacting partners, which ultimately regulates protein function. 14-3-3 proteins serve as prototype for a novel class of scaffold modules that recognize phosphor-Ser/Thr (pS/pT) containing conserved motifs in a variety of proteins. 14-3-3s are highly evolutionarily conserved small, dimeric (homo- and heterodimers) and acidic proteins that are widespread in almost all eukaryotic organisms [3–5]. Whereas yeast has two genetically distinct but structurally homologous isoforms, and mammals seven, most plant genomes contain a dozen 14-3-3 genes which can be divided into two distinct groups: epsilon and non-epsilon, based on their sequence homology and exon/intron structure [3,4,6,7]. Although high degree of sequence similarity among 14-3-3 isoforms suggests functional redundancy, the presence of phenotypes for single and multiple 14-3-3 knock-out mutants suggest that 14-3-3 isoforms selectively bind to their individual protein ligands with different affinities, owing to the spatiotemporal regulation of expression of these isoforms [8–12]. Moreover, large number of 14-3-3 isoforms expressed in an organism suggests high combinatorial complexity in dimer re-arrangement, which in turn fine-tunes their cellular functions.
14-3-3 proteins often interact with their target proteins through canonical phosphorylated motifs, categorized as: Mode I (RXXpS/pT), Mode II (RXXXpS/pT) and Mode III (RXXpS/pTX1–2C′), where X is any amino acid and pS/pT represents phosphoSer or phosphoThr [13–18]. The interaction is mediated by conserved amphipathic groove harbored by 14-3-3 [19,20]. Docking site for 14-3-3 is created by a combination of inherent shape and phosphate-group added in the target protein, in response to a particular signaling cascade . In this context, the stability of 14-3-3 has been put forward as a basis for ‘molecular anvil hypothesis’ in which the rigid 14-3-3 dimer mechanically imposes structural rearrangements in its target phosphorylated protein, thus regulating its functional properties, while itself undergoing only minimal structural alterations . Depending on biochemical nature of the phosphorylated target protein(s), physical association with 14-3-3 can have different functional consequences, resulting either in modulation of their enzymatic activities, subcellular localization, stability or macromolecular interactions [4,11,23–28].
14-3-3 proteins, thus, operate as multipurpose conformation (allosteric) clamps which are recruited to hold their phosphorylated protein partner(s) in place, in response to cellular signaling pathways. The interaction with 14-3-3 culminates in the regulation of cellular functions like apoptosis, adhesion-dependent integrin signaling, cell cycle control in response to genotoxic stress, ion-channels functioning, etc., thus, governing diverse physiological processes and cellular status [4,24–27]. Studies on 14-3-3/client-protein interactions, by utilizing various biochemical and biophysical approaches, may therefore, provide tremendous opportunities for therapeutic interventions under numerous pathological conditions.
In the malaria parasite, P. falciparum, two 14-3-3 isoforms have been annotated by database curators based on sequence similarity with experimentally annotated orthologs: Pf14-3-3I and Pf14-3-3II (accession numbers: PF3D7_0818200 and PF3D7_1362100, respectively), as documented in Plasmodium Genomic Resource database (PlasmoDB; release 46; updated on 6th Nov., 2019; https://plasmodb.org/plasmo/). In the present study, we confirm the presence of 14-3-3 protein in the malaria parasite. Furthermore, we report that Pf14-3-3I interacts with a highly expressed protein in the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) that plays key role in multitude of essential cellular processes, including parasite invasion and egress during intra-erythrocytic proliferative stages of the parasite. Antagonizing the Pf14-3-3I/PfCDPK1 protein–protein interaction (PPI), by using well characterized 14-3-3 recognition motifs, inhibits parasite growth in vitro, which indicates crucial physiological role of this PPI in the parasite. Our work sets a precedent for the rational design of 14-3-3 based PPI inhibitors, by utilizing 14-3-3 recognition motif peptides as a potential antimalarial strategy.
Materials and methods
Unless stated, all materials were purchased from Sigma–Aldrich, St Louis, MO, U.S.A.
Two different phosphopeptides: Mode I (ARSHpSYPA or peptide 1) and Mode II (RLYHpSLPA or peptide 2) (pS: phosphoserine) were utilized to antagonize Pf14-3-3/PfCDPK1 PPI inhibition. These phosphopeptides were identified by Rittinger et al.  from oriented peptide library screening and are recognized by all mammalian and S. cerevisiae 14-3-3 isotypes. The peptides (HPLC purity >98%) were synthesized from Biochain Incorporated, India. The HPLC and Mass spectrometry analysis reports for both peptides are provided in Supplementary File S2.
P. falciparum 3D7
Cryopreserved P. falciparum parasites (3D7 laboratory strain) were thawed and cultured according to the protocol as described by Trager and Jensen . Briefly, parasite cultures were maintained in O+ erythrocytes at 2% hematocrit level, in RPMI 1640 medium (Gibco®, U.S.A.) supplemented with 0.5% AlbuMAXTM I (Gibco®, U.S.A.), 50 mg/L hypoxanthine, 10 mg/L gentamycin (Gibco®, U.S.A.) and 2 gm/L sodium bicarbonate. Parasite culture was maintained in ambient hypoxic environment (5% O2 and 5% CO2; balanced with N2). Prior to setting up an experiment, parasite culture was tightly synchronized at ring stage by lysing erythrocytes parasitized with mature parasites, with 5% sorbitol, for two successive intra-erythrocytic proliferative cycles of the parasite.
Comparative sequence analysis and domain architecture of
Multiple Sequence Alignment (MSA) was performed on comprehensive ensembles of 14-3-3 protein sequences for contextualizing the patterns of conservation and correlation in Pf14-3-3I protein, in light of the structurally and functionally well-characterized 14-3-3 orthologs in humans. Amino acid sequences of 14-3-3 isoforms from Homo sapiens (seven isoforms: 14-3-3 epsilon, P62258; beta/alpha, P31946; zeta/delta, P63104; theta, P27348; gamma, P61981; eta, Q04917 and sigma, P31947) and P. falciparum strain 3D7 (two isoforms: Pf14-3-3I, PF3D7_0818200 and Pf14-3-3II, PF3D7_1362100) were retrieved from Universal Protein Resource Knowledgebase (UniProtKB; https://www.uniprot.org/) and PlasmoDB, respectively [31,32]. MSA was performed by using MultAlin tool, which creates sequence (either protein or nucleic acid) alignment from a group of related sequences by using progressive pairwise alignments. (http://multalin.toulouse.inra.fr/multalin/) . Based on literature review, and comparative sequence analysis with the Hs14-3-3 isoforms, probable amino acid residues of Pf14-3-3I involved in dimerization and phosphopeptide (target) binding were employed to illustrate overall Pf14-3-3I architecture [25,29,34–36]. This was done by using Illustrator for Biological Sequences (IBS 1.0.3), a tool for visualizing biological sequences (http://ibs.biocuckoo.org/) .
Phylogenetic analysis and 14-3-3 binding consensus motifs
Phylogenetic relationship of Pf14-3-3 isoforms with their orthologs present across all three kingdoms of life: animalia, plantae and fungi, was established. To obtain full-length 14-3-3 protein sequences, BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search was performed by using amino acid sequence of Pf14-3-3I protein as query sequence, and whole proteomes of metazoans (taxid: 33208), embryophytes (taxid:3193) and fungi (taxid:4751) as search sets. Publicly available databases such as UniprotKB and The Arabidopsis Information Resource (https://www.arabidopsis.org/) were utilized to retrieve 14-3-3 protein sequences from Homo sapiens and Arabidopsis thaliana, respectively [31,38]. Literature survey was done to identify 14-3-3 proteins from Saccharomyces cerevisiae, and certain plant species including Solanum lycopersicum, Populus trichocarpa, Oryza sativa, Glycine max and Medicago trucatula [39–41]. 14-3-3 protein sequences, thus retrieved, from the BLASTp search, databases and literature survey (Supplementary File S1) were further categorized into two groups: epsilon and non-epsilon. Unrooted phylogenetic relationship of Pf14-3-3 isoforms with these orthologs was established by using MEGA6, a user-friendly software suite for analyzing DNA and protein sequence data from species and populations (http://www.megasoftware.net/) .
To identify experimentally validated 14-3-3 interacting proteins from prokaryotes and eukaryotes, and further collate details of their 14-3-3 binding phosphoSer/Thr sites, literature survey followed by mining of publically available databases was done. The Eukaryotic Linear Motif resource (ELM), a database of experimentally validated eukaryotic linear motifs (http://elm.eu.org/), and ANnotation and Integrated Analysis of the 14-3-3 interactome database (ANIA) which integrates multiple data sets on 14-3-3 binding phosphoproteins (https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py) were used for this purpose [43,44]. The repertoire of experimentally determined gold-standard 14-3-3 binding phosphosites was further extended from the literature survey [45–47]. Amino acid sequences of these 14-3-3 binding phosphopeptides were used to update consensus 14-3-3 recognition motifs by using WebLogo 3, a web based application designed to generate sequence logos (http://weblogo.berkeley.edu/) . Key protein kinases of the parasite (CDPK1, PKG, PKAR and PKAC) were filtered out as putative binding partners of Pf14-3-3I by combining 14-3-3 binding motifs search and global phosphoproteomic datasets of pS/pT peptides enriched from schizont stage of P. falciparum [49–53]. Overall domain architectures of the protein kinases were drawn by using IBS 1.0.3 .
cDNA preparation, molecular cloning, overexpression and purification of recombinant
Pf14-3-3I and PfCDPK1
cDNA from P. falciparum schizonts was prepared by following a previously described protocol, in the absence and presence of Reverse Transcriptase (RT) . Integrity of cDNA, thus obtained, was checked by amplification of transcript encoding for 18s rRNA using the following primer sets: 18s_Fwd, 5′-CCGCCCGTCGCTCCTACCG-3′ and 18s_Rev, 5′-CCTTGTTACGACTTCTCCTTCC-3′. 18s rRNA was also amplified from genomic DNA (gDNA) of the parasite, as positive control. Presence of Pf14-3-3I transcripts in the cDNA pool was confirmed by amplification using the following primer sets: pf14-3-3I_Fwd, 5′-ATGGCAACATCTGAAGAATTAAAACA-3′ and pf14-3-3I_Rev, 5′-TTCTAATCCTTCGTCTTTTGATTTT-3′. pf14-3-3I gene was also amplified from gDNA of the parasite, as positive control. The experiment was done twice. Transcript encoding full-length Pf14-3-3I protein was amplified using the following primer sets: Pf14-3-3I_Fwd, 5′-TGCGGGATCCATGGCAACATCTGAAGAATTAAAACA-3′ and Pf14-3-3I_Rev, 5′-ATTTGTCGACTCATTCTAATCCTTCGTCTTTTGATT-3′, by using Phusion high-fidelity DNA polymerase (Thermo Scientific). Amplified DNA fragment encoding Pf14-3-3I was cloned between BamHI and SalI restriction sites of the expression vector pGEX-4T-1 vector (with a cleavable Glutathione S-Transferase tag at the N-terminus). Recombinant plasmid was transformed into Escherichia coli DH5α competent cells, and positive clones were confirmed by restriction digestion and Sanger sequencing of insert DNA fragment.
For overexpression of GST-Pf14-3-3I fusion protein, the recombinant plasmid containing pf14-3-3I gene was transformed into E. coli strain Rosetta competent cells. Overexpression of the protein was induced with 0.5 mM IsoPropyl β-d-1-ThioGalactopyranoside (IPTG) at 0.4–0.5 Optical Density (OD; at 600 nm) of bacterial secondary culture, for 8 h at 25°C. Cells were harvested by centrifugation at 4000g in Sorvall® EvolutionTM RC Centrifuge (Thermo Fisher Scientific ™) at 4°C for 15 min. Bacterial pellet, thus obtained, was resuspended in cell lysis buffer (buffer A) containing 10 mM HEPES-NaOH, pH 7.4; 1 mM Ethylene Diamine Tetraacetic Acid (EDTA); 150 mM NaCl; 25 µg/ml lysozyme; 3 mM β-MercaptoEthanol (βME), Protease Inhibitor Cocktail (PIC, Roche) and 1 mM PhenylMethylSulfonyl Fluoride (PMSF), followed by sonication for 15 min with successive pulses of 6 s ON and 10 s OFF. Cell lysate was centrifuged at 15,000 g, at 4°C for 1 h. Supernatant, thus obtained, was loaded onto Glutathione Sepharose High Performance resin packed column, GSTrapTM HP 1 ml (GE Healthcare) pre-equilibrated with buffer B (10 mM HEPES-NaOH, pH 7.4; 1 mM EDTA and 150 mM NaCl), for overnight at 4°C. After protein binding, the column was washed with 10 fold column volume of buffer B and recombinant 14-3-3I protein was eluted with different concentrations (10, 20 and 50 mM) of reduced glutathione, prepared in buffer B. Elution fractions, thus obtained, from affinity purification were pooled and buffer exchanged using AmiconTM Ultra-15 Centrifugal Filter Unit (10 kDa cutoff; MerckTM) in 10 mM HEPES-NaOH buffer (pH 7.4) and 150 mM NaCl.
To raise specific antibodies against r14-3-3I protein, four female Balb/c mice (6 to 8 weeks old) were each administered (i.p.) with 50 µg of the recombinant protein (in 0.9% saline), in a prime and boost regimen. For priming dose (day 0), the formulation was made by thoroughly mixing equal volumes of Freund's complete adjuvant and saline containing the protein. For subsequent booster doses (days 21 and 42), formulations were made with Freund's incomplete adjuvant. Blood samples were collected from retro-orbital sinus of the mice on days 31 and 52 (terminal bleed) after primary immunization. Collected blood samples were incubated for 30 min at 37°C, centrifuged at 1200×g at 4°C for 15 min, and then serum samples were collected and stored at −80°C until further analysis. Once the antiserum was raised, native Pf14-3-3I protein was detected in schizonts lysate by immunoblotting. The experiment was done twice.
Cloning of pfcdpk1 gene in pET-28a(+) vector (with 6x-His tags at N- and C-termini) was done as described earlier . For overexpression of 6x-His-PfCDPK1 fusion protein, the recombinant plasmid harboring pfcdpk1 gene was transformed into E. coli strain BLR (λDE3) competent cells. Overexpression of the protein was induced with 1 mM IPTG at 0.9 OD of bacterial secondary culture, for 4 h at 30°C. Protein purification was done in a similar manner as described above for the purification of r14-3-3I, except for the following changes in buffers’ compositions: buffer A: 10 mM Tris, pH 8.0; 1 mM EDTA; 100 mM NaCl; 25 µg/ml lysozyme; 3 mM βME and PIC; and, buffer B: 10 mM imidazole; 10 mM Tris, pH 8.0; 100 mM NaCl and 3 mM βME. Supernatant obtained after centrifugation of bacterial lysate was loaded onto High-Performance Immobilized Metal Affinity Chromatography (IMAC) column, HisTrapTM HP 1 ml (GE Healthcare) and rCDPK1 protein was eluted with different concentrations of imidazole ranging from 20–500 mM, prepared in buffer B. Elution fractions, thus obtained, from metal-affinity purification were pooled and buffer exchanged, as mentioned above for r14-3-3I protein.
Immuno-Fluorescence assay (IFA) to detect expression and co-localization of
Pf14-3-3I with PfCDPK1
IFAs were performed on synchronized P. falciparum 3D7 culture to check for expression of Pf14-3-3I and its co-localization with PfCDPK1 in mature stages of the parasite, as described earlier . Briefly, mature schizonts (44–46 hpi) and merozoites were isolated, smeared on glass slides, dried and fixed with pre-chilled methanol for overnight at −20°C. Non-specific binding sites in the parasite were blocked with 3% BSA (in 1× PBS), for 1 h at room temperature (RT, 293 K) and probed with anti-Pf14-3-3I mouse serum and anti-PfCDPK1 rabbit serum at dilutions of 1 : 50, for 1 h at RT. Slides were washed twice with PBS containing 0.05% Tween-20 (PBST), followed by washing once with PBS, and probed with Alexa-Fluor 488 conjugated anti-mouse IgG (Molecular Probes, U.S.A.) and Alexa-Fluor 594 conjugated anti-rabbit IgG, both at dilutions of 1 : 500, for 1 h at RT. All antibodies were diluted in 1% BSA, prepared in 1X PBS. The slides were washed and mounted with ProLong Gold antifade reagent with DAPI (4′,6- diamidino-2-phenylindole; Invitrogen). Images were acquired in Olympus FLUOVIEW FV3000 confocal microscope using Olympus cellSens dimensions imaging software. Expression and Co-localization analysis from all three channels (blue: DAPI; green: Pf14-3-3I; red: PfCDPK1) was performed on a pixel by pixel basis using the same software. Every pixel in the image was plotted in the scatter diagram based on its intensity level from the channels, followed by estimation of Pearson's correlation coefficient, r. For the estimation of ‘r’, an infected and stained RBC was selected using the Region of Interest (ROI) tool provided in the software. From this ROI area, the ‘r’ value was noted as given by the software in ROI statistics . The experiment was done thrice.
Homology modeling of
Pf14-3-3I and PfCDPK1
Three-dimensional structure of a protein can provide us with precise information about its single, most stable conformation, as dictated by its sequence. Comparative or homology modeling, one of the most common structure prediction methods in structural genomics and proteomics, was employed to model 3D structures of Pf14-3-3Idimer and PfCDPK1 from P. falciparum strain 3D7. To accomplish this feat, amino acid sequences of Pf14-3-3I (PF3D7_0818200) and PfCDPK1 (PF3D7_0217500) were retrieved from PlasmoDB database . To search for a suitable template for homology modeling, BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search was performed by using amino acid sequences of Pf14-3-3I and PfCDPK1 as query sequences, against Protein Data Bank (PDB) database (http://www.rcsb.org/) [56,57]. Amino acid sequence identity between 14-3-3 orthologs from P. falciparum strain 3D7 (14-3-3I) and Homo sapiens (14-3-3 Epsilon) was found to be 63.52%, thus, rendering X-Ray diffraction based structural model of Hs14-3-3 epsilon (PDB ID: 3UAL; resolution: 1.8 Å) as a suitable template to model 3D structure of Pf14-3-3Idimer . For PfCDPK1, X-ray diffraction based structural model of CDPK1 protein from P. berghei strain ANKA (PDB ID: 3Q5I; sequence similarity: 93%) was used as a suitable template, as described previously by our group [59,60]. Homology modeling was done by using Modeller v9.17 software (https://salilab.org/modeller/), a program designed for comparative protein structure modeling by satisfaction of spatial restraints . The best structural model with the most negative DOPE score was selected and rendered with PyMOL Molecular Graphics System, v2.1 by Schrödinger, LLC (http://pymol.org/2/) .
Generated models were further subjected to structural refinement by using ModRefiner (https://zhanglab.ccmb.med.umich.edu/ModRefiner/) which is an algorithm-based approach for atomic-level, high-resolution protein structure refinement . Reliability of the refined structural models of Pf14-3-3Idimer and PfCDPK1 was assessed by examining backbone dihedral (torsion) angles: phi (Ø) and psi (Ψ) of the amino acid residues lying in the energetically favorable regions of Ramachandran space . This was done by using PROCHECK v.3.5 (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/) . Percent quality measurement of the protein structures was evaluated by using four sorts of occupancies called ‘core’, ‘additional allowed’, ‘generously allowed’ and ‘disallowed’ regions. The refined 3D structural models of Pf14-3-3Idimer and PfCDPK1, thus generated, were subsequently used for Pf14-3-3Idimer/PfCDPK1 PPI and Pf14-3-3Idimer/peptides interaction, and molecular dynamics simulation studies.
Molecular docking of the structurally modeled phosphopeptides (peptide 1: ARSHpSYPA and peptide 2: RLYHpSLPA; pS: phosphorylated serine) against Pf14-3-3Idimer was performed by using AutoDock 4.2.6 tool (http://autodock.scripps.edu/) . For the receptor Pf14-3-3Idimer protein, we assigned polar Hydrogen and Kollman charges, whereas Marsilli-Gasteiger partial charges were assigned for the peptides. We have not used any additional restraint on torsion angles of the receptor protein during the docking process. The Lamarckian Genetic Algorithm (LGA) was used for performing the energy evaluations with a default set of docking parameters. All the docked complexes were evaluated based on their lowest binding energy (kcal/mol).
PfCDPK1 was docked against Pf14-3-3Idimer receptor by using High Ambiguity Driven biomolecular DOCKing (HADDOCK) webserver, an information-driven flexible docking approach for the modeling of biomolecular complexes . We preferentially selected those Ser/Thr residues that were previously reported to be autophosphorylated by PfCDPK1 . To phosphorylate them in silico, we adopted the PyTMs plugin in PyMOL Molecular Graphics System . During the whole docking process, the phosphorylated Ser/Thr residues present at the interface of pCDPK1 and Pf14-3-3Idimer receptor were considered as active residues, and amino acid residues surrounding those as passive residues.
Molecular dynamics (MD) simulation
Preparation and parameterization
Pf14-3-3Idimer/pCDPK1 and Pf14-3-3Idimer/peptides complex systems were parameterized by using terminal interface, tLeap of Assisted Model Building with Energy Refinement tool, AMBER 14 . The system was neutralized by using the counter-charged ions (Na+ or Cl−) followed by solvation using explicit TIP3P water model spanning for 12 Å around the complex systems. The Forcefield ff03.r1 was used for the proteins and peptides, and the phosphorylated Ser and Thr residues were handled by using the ffptm force filed [71,72].
Pf14-3-3Idimer/pCDPK1 and Pf14-3-3Idimer/peptides complex systems were equilibrated by carrying out a short minimization (1000 steps, 500 cycles of steepest descent and 500 cycles of conjugate gradient minimization) runs followed by 50 ps of heating, 50 ps of density equilibration and 500 ps of constant pressure equilibration, performed at 300 K. During the equilibration process, the bonds involving Hydrogen-atoms were handled by SHAKE algorithm . The simulations were carried out at 1 fs time step with Langevin thermostat, under the ‘periodic boundary conditions' using sander, an AMBER 14 module which carries out energy minimization, molecular dynamics, and NMR refinements.
After the equilibration, production runs were performed at 300 K with Langevin thermostat, under a constant pressure. We performed 20 ns long simulations for Pf14-3-3Idimer/peptides complexes and 100 ns long simulation for Pf14-3-3Idimer/pCDPK1 complex, and the co-ordinates were updated after every 5 ps. We used a non-bonded cutoff distance of 8 Å, and Particle Mesh Ewald (PME) algorithm handled the long ranged electrostatic interactions . The net translational and rotational velocities were canceled after every 1000 steps. The simulations were carried out at 1 fs time step by using pmemd, a version of sander that is optimized for speed and parallel scaling.
Analysis and binding energy calculation
PfCDPK1 in vitro
PfCDPK1 phosphorylation was achieved by setting up a kinase reaction in vitro with 100 ng/µl of rCDPK1 in assay buffer [100 mM Tris–Cl, pH 7.4; 2.5 mM DiThioThreitol (DTT); 50 mM MgCl2 and 2.5 mM MnCl2]. Enzymatic reaction was carried out in a total volume of 200 µl, in the absence and presence of Ca2+-ions for conditions requiring minimally (represented as rCDPK1) and maximally (represented as rpCDPK1) phosphorylated PfCDPK1 protein, respectively. An amount of 2.5 mM EGTA was added for condition requiring absence of Ca2+-ions. Kinase reactions were initiated by adding 1 mM ATP and allowed to take place at 30°C for 1 h. Auto-phosphorylation status of PfCDPK1 was confirmed by in-gel fluorescence staining with phosphoamino acid stain, Pro-Q Diamond (Thermo Fisher Scientific™). Protocol was followed as recommended by the supplier. Briefly, SDS–PAGE was performed for the phosphorylation reactions carried out in the absence and presence of Ca2+-ions. 10 µg each of rCDPK1 and rpCDPK1 was loaded onto gel lanes. Phosphostained gels were imaged using Molecular Imager® Pharos FXTM Systems (Bio-Rad). Excitation was set at 555 nm with emission filter of 580 BP 30. Estimates of band intensities of rCDPK1 and rpCDPK1 from phosphostained gel images were made using ImageJ 1.52a, which is a public domain Java image processing program. Lanes and bands were auto-detected and manually size-adjusted to ensure full band inclusion and equivalent thickness across all lanes. In all subsequent experiments, minimally and maximally phosphorylated PfCDPK1 protein is represented as rCDPK1 and rpCDPK1, respectively.
PfCDPK1 enzymatic activity in vitro
Phosphorylation experiments were performed with 100 ng of rCDPK1, per reaction, in assay buffer (100 mM Tris–Cl, pH 7.4; 2.5 mM DTT; 50 mM MgCl2 and 2.5 mM MnCl2) by following protocol, as described previously by our group [60,77]. Enzymatic reaction was carried out in the absence and presence of Ca2+-ions. An amount of 10 µg of dephosphorylated casein from bovine milk, per reaction, was used as exogenous substrate for the enzyme. An amount of 2.5 mM EGTA was added for conditions requiring absence of Ca2+ ions. Kinase reactions were initiated by adding 1 mM ATP and allowed to take place at 30°C for 1 h. To test for any regulatory effect of Pf14-3-3I on the functional activity of PfCDPK1, rCDPK1 was allowed to pre-incubate with equivalent amount of r14-3-3I in the reaction buffer, at room temperature for 1 h, followed by initiating the reaction with the addition of ATP. After completion of the reactions, phosphorylation status of PfCDPK1 and casein was checked by in-gel fluorescence staining with phosphoamino acid stain, Pro-Q Diamond (Thermo Fisher Scientific™). Protocol was followed as described above to confirm autophosphorylation of CDPK1. The experiment was done twice.
ELISA to confirm
Pf14-3-3I/ PfCDPK1 interaction
ELISA was performed to confirm Pf14-3-3/PfCDPK1 interaction, by following previously described protocol . Briefly, purified PfCDPK1 protein (200 ng/100 µl/well of rCDPK1 or rpCDPK1) was coated onto Poly-l-Lysine treated 96-welled microtitre plate, in 0.06 M carbonate-bicarbonate buffer (pH 9.6), for overnight at 4°C. The plate was blocked with 5% skimmed milk solution (in PBS; 200 µl/well) for 1 h at 37°C and washed thrice with 0.05% Tween in PBS (PBST) for 5 min each. For interaction analysis, PfCDPK1 coated wells were incubated with different concentrations of r14-3-3I protein: 0.5, 1, 2, 4 and 8 µM (100 µl/well), for 1 h at 37°C. Equivalent concentrations of GST were taken as negative controls. Following three washes with PBST, anti-GST HRP Conjugated antibody (Sigma–Aldrich; diluted 1 : 5000 in PBS) was added to each well (100 µl/well) and incubated for 1 h at 37°C. The peroxidase reaction was developed with o-Phenylenediamine Dihydrochloride (OPD; 1 mg/ml) as chromogen and hydrogen peroxide as substrate, both prepared in citrate phosphate buffer, pH 5.0 (100 µl/well). The enzymatic reaction was stopped with 2N H2SO4 and the optical density was measured spectrophotometrically by taking absorbance at 490 nm using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific™). For conditions requiring presence of peptides (1 or 2) as Pf14-3-3I/PfCDPK1 interaction inhibitor, ELISA was performed in the presence of different concentrations of each peptide: 1, 5, 10, 25 and 50 µM. The experiment was done twice in triplicates.
Pull-down assay to confirm
Pf14-3-3I/ PfCDPK1 interaction
To further confirm Pf14-3-3I/PfCDPK1 interaction, western blot based GST pull-down assay was performed in which Glutathione Sepharose® 4B beads (GE Healthcare) were coupled with 10 µg of r14-3-3I in buffer A (20 mM Tris, pH 7.4; 0.5 mM DTT; 10 mM MgCl2; 0.5 mM MnCl2; 50 mM NaCl; 4% (v/v) glycerol; 0.1 mg/ml BSA and 25 mM CaCl2), for 1 h at RT, followed by washing three times (at 900 rpm for 2 min.) with the same buffer. Equivalent amount of GST was taken as negative control. PfCDPK1 was auto-phosphorylated in an in vitro kinase reaction, as mentioned above. r14-3-3I bound beads were incubated with PfCDPK1 (10 µg of rCDPK1 or rpCDPK1, per binding reaction), for 1 h at RT. After washing three times with buffer B (buffer A with 300 mM NaCl), bead-bound protein complexes were boiled in SDS-loading dye and analyzed by SDS–PAGE, followed by immunoblotting with HRP-conjugated anti-His antibody (Sigma–Aldrich) by using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific). The blot was stripped and re-probed with HRP-conjugated anti-GST antibody to check for equal coupling of r14-3-3I (or GST) with the beads in all binding reactions. For conditions requiring presence of peptides (1 or 2) as Pf14-3-3I/PfCDPK1 interaction inhibitor, GST pull-down assay was performed in the presence of 10 µM concentration of each peptide. Two independent experiments were performed.
Surface plasmon resonance analysis of binding affinity between
PfCDPK1 and Pf14-3-3I
rCDPK1 protein was auto-phosphorylated in an in vitro kinase reaction, as mentioned above. To determine binding strength of PfCDPK1/Pf14-3-3I complex, real-time biomolecular interaction analysis with Surface Plasmon Resonance (SPR) was carried out at physiologically relevant concentrations, by using AutoLab Esprit SPR (at Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi, India). Kinetic rate constants: Ka and Kd (association and dissociation rate constants, respectively) as well as affinity constant, KD were measured at RT. SPR analysis was performed by following previously described protocols [60,79–83]. Briefly, 5 µM of r14-3-3I was immobilized on the surface (self-assembled monolayer of 11-Mercapto-Undecanoic Acid, MUA on gold surface) of SPR sensor chip by the mechanism of covalent amine coupling. The chemistry involves covalent linkage of esterified carboxyl groups of the sensor surface with ε-amino group of lysine residues of a given protein. Unreacted ester groups on the sensor chip surface were blocked with 100 mM Ethanolamine, pH 8.5. Interaction kinetics was studied by injecting rCDPK1 or rpCDPK1 over the r14-3-3I immobilized chip surface at different concentrations: 100, 250, 500, 750 and 1000 nM, at a steady rate of 20 µl/min, with association and dissociation time of 300 s and 150 s, respectively. HEPES-NaOH buffer was used both as immobilization and binding solution. Surface of the sensor chip was regenerated with 50 mM NaOH. The experiment was done twice.
Isothermal titration calorimetric analysis
Evaluation of growth inhibitory effect on malaria parasites
In the bar graphs, data is expressed as Mean ± Standard Deviation (SD) of three independent experiments, done in duplicates. Statistical analysis was done by using OriginPro Evaluation 2018b Graphing and Analysis software. Unless indicated, the differences were considered to be statistically significant at P < 0.05; ns (not significant) indicates P > 0.05, one star indicates P < 0.05 and two stars indicate P < 0.01.
Sequence analysis and identification of
MultAlin-based sequence alignment of Pf14-3-3I with its well-characterized orthologs in humans demonstrated patterns of conservation and correlation in Pf14-3-3I (Figure 1a). Residues with high consensus value (>90%) are shaded in red and those with low consensus value (<90% and >50%) are shaded in blue. α-helices and Nuclear Export Signal (NES) are also indicated. Five highly conserved sequence blocks, as identified by Wang and Shakes  were observed, as shown boxed and shaded red. Residues at the dimerization interface (solid circles) and residues involved in phosphopeptide (target) binding (solid squares) were found to be conserved in all 14-3-3 isotypes, except Pf14-3-3II which appears to be the most divergent form of 14-3-3 proteins. Based on the literature review [25,29,34–36] and comparative sequence analysis with the Hs14-3-3 isoforms, probable amino acid residues of Pf14-3-3I involved in dimerization and phosphopeptide (target) binding were identified and highlighted in the overall Pf14-3-3I architecture (Figure 1b).
Comparative sequence analysis, protein architecture and identification of
P. falciparum 14-3-3I.
X-Ray diffraction based structural model of Hs14-3-3 epsilondimer was used as a template to generate three-dimensional co-ordinates of Pf14-3-3Idimer. After optimal rigid-body superimposition of the generated structural model of Pf14-3-3Idimer with Hs14-3-3 epsilondimer, overall Root-Mean-Square Deviation (RMSD) value of the C-alpha atomic co-ordinates was found to be 0.63 Å, suggesting a reliable 3D structure. Pf14-3-3Idimer homology model revealed strong resemblance with it's counterparts from other living organisms, with overall folds forming a clamp like structure, where each monomer is capable of forming a functional amphipathic groove for binding to phosphorylated residues (pS/pT) on target protein(s). Additionally, both monomers of Pf14-3-3Idimer were found to be oriented in opposite direction with respect to each other. Helical regions in one of the monomers of Pf14-3-3dimer are marked from H1 to H9 (Supplementary Figure S1a). Assessment of stereochemical quality and accuracy of the homology model displayed 88.7% of amino acid residues lying in the most favored (core) regions, with 8.8%, 1.5%, and 1.1% residues in additional allowed, generously allowed and disallowed regions of Ramachandran plot, respectively (Supplementary Figure S1b). The comparable RMSD value and Ramachandran plot characteristics confirmed the reliability of the 3D-structural model of Pf14-3-3Idimer to be taken further for docking and simulation studies.
Unrooted phylogenetic relationship of Pf14-3-3 isoforms with their orthologs present across three major kingdoms of life: plantae, animalia and fungi showed that Pf14-3-3 isoforms have followed convergent evolutionary pathway with 14-3-3 proteins from plant non-epsilon group (Supplementary Figure S2a). Branches with green, red and blue squares represent 14-3-3 proteins from kingdom plantae, animalia and fungi, respectively. Detailed evolutionary relationship of Pf14-3-3 isoforms with their orthologs from plant non-epsilon group is shown in Supplementary Figure S2b.
Transcript encoding for Pf14-3-3I protein was amplified from cDNA prepared from schizonts, by using pf14-3-3I specific primers, as described in materials and methods section. Detection of desired DNA fragment of 789 bp indicated the presence of Pf14-3-3I encoding cDNA in the parasite, as compared with the band size of 1449 bp using gDNA as template (Figure 1c). Amplification of transcript encoding for 18s rRNA (120 bp) by using pf18s specific primers was taken as positive control. Furthermore, western blot analysis of native Pf14-3-3I protein in the parasite (schizonts) lysate, by using in-house generated polyclonal mice sera raised against r14-3-3I, confirmed the existence of Pf14-3-3I protein in the parasite (Figure 1d). Desired protein band of ∼30.2 kDa was observed in both Cytosolic (C) and Membrane (M) fractions of the parasite lysate. IFA in mature schizonts and free merozoites with the anti-r14-3-3I mice serum indicated localization of the protein towards cell periphery (Figure 1e (i)). In support of the confocal images, localization analysis is shown by a representative scatter plot (Figure 1e (ii)) and mean Pearson's correlation coefficient (r) (Figure 1e (iii)), as analyzed by using Olympus cellSens dimensions imaging software. Green intensity (Pf14-3-3I) is shown on the x-axis and blue intensity (DAPI) is shown on the y-axis; and, r = 0.25 ± 0.12 (n = 34).
To update consensus 14-3-3 recognition motifs, literature search followed by mining of publically available databases was done to identify experimentally validated 14-3-3 interacting proteins present in prokaryotes and eukaryotes, and further collate details of 14-3-3 binding phosphoSer/Thr sites in the target proteins. In total, 323 Mode I sites from 243 target proteins, 81 Mode II sites from 77 target proteins and 9 Mode III sites from 9 target proteins were identified as gold-standard 14-3-3 binding pS/pT sites (Supplementary Table S1). Amino acid sequences of these 14-3-3 binding phosphopeptides were used to update consensus 14-3-3 binding motifs, as shown in Supplementary Figure S3a. Furthermore, key protein kinases of the parasite: PfCDPK1, Protein Kinase G (PfPKG), Protein Kinase A regulatory subunit (PfPKAR) and Protein Kinase A catalytic subunit (PfPKAC) were filtered out as putative binding partners of Pf14-3-3I, by combining global phosphoproteomic datasets of peptides enriched from schizont stage of P. falciparum, with 14-3-3 binding motifs search (Supplementary Figure S3b).
Molecular Dynamics (MD) simulation reveals stable
Pf14-3-3Idimer/ pCDPK1 complex formation
To probe the associated inter-molecular interactions that regulate Pf14-3-3Idimer/pCDPK1 binding affinity, we performed MD simulation studies. A schematic representation of the interactions between Pf14-3-3Idimer and pCDPK1 is shown in Figure 2a. Peptide stretch, 61KLGS64 of pCDPK1 was found to play a key role in mediating its interaction with Pf14-3-3Idimer, where a Hydrogen-bond was formed between S64 of pCDPK1 and K227 of Pf14-3-3Idimer, with a bond length of 2.92 Å. In addition to the Hydrogen-bond, inter-molecular association was further stabilized by hydrophobic interactions between S64 of pCDPK1 and Y226, Y555, E557, I597 constituting amphipathic groove of Pf14-3-3Idimer. Furthermore, comprehensive analysis of the MD trajectories, i.e. Root Mean Square Deviation (RMSD), Radius of Gyration and variation in the Hydrogen-bond formation between Pf14-3-3Idimer and pCDPK1 were also reported as a function of simulation time (Figure 2a). The variation of RMSD and Radius of Gyration in Pf14-3-3Idimer and pCDPK1 clearly indicated a lesser stability and compactness in the former compared with the latter. Stability of the Pf14-3-3Idimer/pCDPK1 complex was also confirmed by the extensive Hydrogen-bond formation between the two proteins, at different time intervals throughout the simulation run, and a negative binding free energy of −36.8 ± 1.0 kcal/mol, as obtained from MM/GBSA method. Movie S1 shows the interactions between Pf14-3-3dimer (blue) and pCDPK1 (grey).
Molecular dynamics simulation of
Pf14-3-3/ pCDPK1 complex.
Amino acid sequences of probable Pf14-3-3 binding phosphopeptides of PfCDPK1 are shown in Figure 2b. These peptide stretches were identified by combining global phosphoproteomic datasets of peptides enriched from schizont stage of P. falciparum, with 14-3-3 binding motifs search.
Pf14-3-3I exhibits divergent binding affinities for CDPK1 and pCDPK1 in vitro
Pf14-3-3I interacts with PfCDPK1 in vitro.
For ELISA based Pf14-3-3I/PfCDPK1 PPI analysis, poly-l-Lysine treated 96-welled microtitre plate was coated with the bait protein, rCDPK1 (or rpCDPK1), followed by addition of increasing concentrations of the prey protein, r14-3-3I (or GST, as negative control). The interaction analysis was done by using monoclonal antibody against GST protein. Concentration dependent binding between rCDPK1 (or rpCDPK1) and r14-3-3I was observed (Figure 3b). Furthermore, PPI analysis by ELISA indicated that phosphorylation status of PfCDPK1 dictates its interaction with r14-3-3I. No significant absorbance (OD490 nm) was observed for interaction of rCDPK1 (or, rpCDPK1) with GST alone. To quantitate the interaction between rCDPK1 (or rpCDPK1) and r14-3-3I, SPR analysis was performed by using AutoLab Esprit SPR. r14-3-3I was immobilized at an average density of 4.3 ng per 1 mm2 of the sensor chip surface. Once immobilized, r14-3-3I demonstrated good stability throughout the experiment. Interaction analysis was done by injecting serial dilutions of either rCDPK1 or rpCDPK1, ranging from 100 nM to 1 µM, over the r14-3-3I-immobilized sensor chip surface, followed by comparing their respective kinetics and binding affinities at RT. With increase in mass concentration of rCDPK1 and rpCDPK1, gradual increase in sensor signal was observed, which linearly correlated with corresponding change in refractive index of the medium immediately adjacent to the SPR sensing surface. Concentration dependent real-time sensorgrams alongwith KD values of the interactions are shown in Figure 3c. rCDPK1 and rpCDPK1 showed differential binding affinities for r14-3-3I, with KD values of 0.67 ± 0.0036 µM and 1.35 ± 0.0083 µM, respectively. To provide mechanistic insight into the role of Pf14-3-3 in regulating PfCDPK1 activity, we tested kinase activity of rCDPK1 in vitro, in the presence of r14-3-3. However, we observed only marginal increment in the phosphorylation status of casein, and no significant increase in auto-phosphorylation status of PfCDPK1, as confirmed by in-gel staining of the reaction mixtures with Pro-Q Diamond phosphoamino acid stain (Figure 3d and Supplementary Figure S4).
IFA was performed on synchronized P. falciparum 3D7 culture to check for co-localization of 14-3-3I and CDPK1, by probing mature stages of the parasite with anti-Pf14-3-3I mouse-serum and anti-PfCDPK1 rabbit-serum. 14-3-3I protein was found to co-localize very nicely with CDPK1 protein towards cell periphery, in mature schizonts and free merozoites (Figure 3e (i)). In support of the confocal images, co-localization analysis is shown by a representative scatter plot (Figure 3e (ii)) and mean Pearson's correlation coefficient (r) (Figure 3e (ii)), as analyzed by using Olympus cellSens dimensions imaging software. Green intensity (Pf14-3-3I) is shown on the x-axis and red intensity (PfCDPK1) is shown on the y-axis; and, r = 0.79 ± 0.04 (n = 34). Yellow dots on the scatter plot show the co-localization of Pf14-3-3I and PfCDPK1.
We utilized two different phosphopeptides: Mode I (ARSHpSYPA or peptide 1) and Mode II (RLYHpSLPA or peptide 2) (pS: phosphoserine) to inhibit Pf14-3-3/PfCDPK1 interaction [87–92], as mentioned in materials and methods section. These phosphopeptides were identified by Rittinger et al.  from oriented peptide library screening, and are recognized by all mammalian and S. cerevisiae 14-3-3 isotypes.
MD simulation reveals stable
Pf14-3-3Idimer/peptides (1 or 2) complex formation
Molecular interactions between Pf14-3-3Idimer and phosphopeptides (1 or 2) were studied by performing 20 ns MD simulations (Figure 4a). Peptide 1 was found to bind in the amphipathic groove of the receptor protein, Pf14-3-3Idimer. Here, three Hydrogen-bonds were formed between phosphoSer of peptide 1 and K306, K379, S302 constituting amphipathic groove of Pf14-3-3Idimer, in addition to hydrophobic interactions that further stabilized the association. Similarly, phosphoSer of peptide 2 formed a Hydrogen-bond with N188 of Pf14-3-3Idimer, along with stabilizing hydrophobic interactions. A comparative study using time-dependent variations in RMSD and Radius of Gyration, however, revealed that Pf14-3-3Idimer/peptide 2 complex was more stable and slightly more compact compared with Pf14-3-3Idimer/peptide 1 complex. The observation was supported by the higher number of inter-molecular Hydrogen-bonds that are formed between Pf14-3-3Idimer and peptide 2, as compared with peptide 1. This also accounts for the higher negative binding free energy observed for Pf14-3-3Idimer/peptide 2 complex (−67.2 ± 0.1 kcal/mol) relative to Pf14-3-3Idimer/peptide 1 complex (−41.8 ± 1.0 kcal/mol). Supplementary Movies S2 and S3 show the interactions of Pf14-3-3dimer (blue) with peptides 1 and 2 (yellow), respectively.
Phosphopeptides 1 and 2 interact with
Phosphopeptides 1 and 2 interact with
Pf14-3-3I in vitro
After affirming the interaction of Pf14-3-3Idimer with phosphopeptides 1 and 2 through MD simulation studies, we further sought to establish the knowledge of binding modes of complexation, by employing ITC. This was achieved by determining binding affinity constant (Ka), and thermodynamic parameters associated with the interaction, like change in enthalpy (ΔH), binding free energy (ΔG) and entropy (ΔS). The representative binding isotherms resulting from titration of peptides (1 or 2) with r14-3-3I are shown in Figure 4b. Binding isotherms for peptide 1 and 2 were sigmoidal in nature ending near zero baselines, indicating saturation of r14-3-3's binding sites. In Figure 4b, solid line shows the best fit of the non-linear experimental data, and the model reproduces experimental data fairly well. On the basis of Ka values, binding strength was found to be higher in case of r14-3-3I/peptide 1 complex (Ka: 1.7 × 106 ± 6.8 × 105 M−1) than that of r14-3-3I/peptide 2 (Ka: 8.3 × 105 ± 2.67 × 105 M−1). For peptides 1 and 2, binding stoichiometry (N) was found to be 0.3–0.4 and ∼1.0, respectively. Moreover, it was observed that the binding of peptide 1 with r14-3-3I protein was enthalpically favorable (ΔH = −11.34 ± 4.27 kcal/mol), whereas entropically unfavorable [TΔS = −2.8 kcal/mol (±15–20%)], resulting in strong binding free energy [ΔG = −8.5 kcal/mol (±15–20%)]. Higher negative ΔH value indicates the ability of peptide 1 to form a strong network of H-bonds and Van Der Waals interactions with Pf14-3-3I protein. The associated unfavorable ΔS value may be the result of loss of conformational freedom upon binding with Pf14-3-3I. For r14-3-3I/peptide 2 complex, the interaction was enthalpically as well as entropically driven [ΔH = −1.52 ± 0.096 kcal/mol, TΔS = 6.5 kcal/mol (±15–20%) and ΔG = −8.0 kcal/mol (±15–20%)]. Higher positive ΔS value suggests contribution from solvation entropy, which results from the loss of water molecules from the binding surfaces of interacting partners when they come close together to form a complex . And, small negative ΔH value indicates smaller number of H-bonds contributing to the formation of Pf14-3-3I/peptide 2 complex.
Phosphopeptides 1 and 2 antagonize
Pf14-3-3I/ PfCDPK1 interaction
ELISA based Pf14-3-3I/PfCDPK1 PPI analysis was performed in the presence of varying concentrations of phosphopeptides (1 or 2). Concentration dependent r14-3-3I/rpCDPK1 interaction inhibition was observed (Figure 5a). To further confirm ELISA results, western blot based GST pull-down assay was performed in which r14-3-3I (or GST, as negative control) was coupled with Glutathione Sepharose® 4B beads, followed by binding with rCDPK1 or rpCDPK1 to form bead-bound protein complexes, in the absence and presence of 10 µM concentration of the peptides. Immunoblotting with HRP-conjugated anti-His antibody indicated that phosphorylation status of PfCDPK1 dictates its interaction with Pf14-3-3I, as confirmed by ELISA (Figure 5b). Moreover, interaction inhibition was readily observed in the presence of peptides (1 or 2). The same blot stripped and re-probed with HRP-conjugated anti-GST antibody confirmed equal coupling of r14-3-3I (or GST) with the beads in all binding reactions.
Phosphopeptides 1 and 2 antagonize
Pf14-3-3I/ PfCDPK1 interaction.
Phosphopeptides 1 and 2 with propensity to bind Pf14-3-3I and inhibit Pf14-3-3I/PfCDPK1 interaction, were further subjected to P. falciparum invasion inhibition in vitro. Towards this, mature (punctated) schizonts were allowed to burst and invade into erythrocytes, in the presence of 12.5 µM concentration of the peptides. Untreated parasites served as control and percentage of parasite invasion was evaluated by flow cytometry, as described in materials and methods section. Peptides 1 and 2 showed significant inhibition in progression of schizonts to ring stage of the parasite (Figure 5c (i)). Giemsa stained microscopic images of mature schizonts incubated with peptides 1 and 2 are shown in Figure 5c (ii). Control parasites were healthy, producing healthy rings at 22 h post-incubation. Whereas, upon treatment with the peptides, merozoite invasion inhibition was observed with significant alteration in parasite morphology. Here, relatively less number of ring parasitized erythrocytes were observed, alongwith the accumulation of pyknotic forms of the parasite.
There are only a few evidences in literature which support the existence of 14-3-3 protein in Plasmodium species. 14-3-3 gene in P. falciparum and P. knowlesi was first identified by Al-Khedery et al. , who reported that pk14-3-3 transcript begins to be expressed in the ring-stage, predominates in young trophozoites, and thereafter declines, during asexual intra-erythrocytic proliferative stages of the parasite. Also, antiserum raised against recombinant Pk14-3-3 was able to cross-react with Pf14-3-3. Later, Lalle et al.  showed that in P. berghei parasitized erythrocytes, the host junctional complex protein, demantin, is repositioned to the parasite where it interacts with Pb14-3-3 in a phosphorylation dependent manner, thus influencing remodeling of the erythrocytic skeleton. Furthermore, Dastidar et al.  identified Pf14-3-3 as a novel member of P. falciparum histone phosphosite binding protein repertoire, isolated from asexual blood stages of the parasite.
Two 14-3-3 isoforms are annotated in P. falciparum by PlasmoDB database curators: Pf14-3-3I and Pf14-3-3II. Upon their comparative sequence analysis with structurally and functionally characterized orthologs from humans, we reported patterns of sequence and structural conservation in Pf14-3-3I protein sequence, particularly encompassing amino acid residues at the dimerization interface and those involved in phosphopeptide binding. Concomitantly, our phylogenetic analysis showed that despite having evolved separately since the early eukaryotes, Pf14-3-3I has followed convergent evolution with plant non-epsilon group. In plants, the epsilon group of 14-3-3 proteins is considered as ancestral and destined to fulfil fundamental cellular functions; whereas, the non-epsilon group evolved later and members of this group might be related to organism specific functions . We further confirmed the presence of 14-3-3 in the malaria parasite by employing various molecular biology techniques. Real-time PCR indicated the presence of transcript encoding for Pf14-3-3I protein. Western blot analysis using in-house generated polyclonal sera raised in mice against recombinant Pf14-3-3I protein indicated the presence of full length protein in the parasite. Furthermore, confocal microscopic imaging in mature schizonts and free merozoites indicated that Pf14-3-3I protein localizes towards the cell periphery.
In metazoans, kinases have been reported to be functionally modulated as a consequence of their interaction with 14-3-3 proteins. In this regard, first report came from Aitken et al. (1990, 1992) who identified 14-3-3 as one of the potent inhibitors of Ca2+-dependent Protein Kinase C (PKC) in sheep brain, where it could inhibit phosphorylation activity of the kinase over a wide range of Ca2+-ion concentrations [98–100]. In the concurrent year, Robinson et al.  investigated the mechanism of action of individual 14-3-3 isoforms in the inhibition of PKC. In the same line, Wheeler-Jones et al.  used synthetic peptides derived from conserved sequences of 14-3-3 that resemble pseudo-substrate regions of PKC and annexins, and suggested that 14-3-3 isoforms play a physiological role as inhibitors of PKC activity in human platelets, but are unlikely to be involved in controlling association of PKC with the membrane. Furthermore, Hausser et al.  identified 14-3-3tau as a negative regulator of PKCµ, in which 14-3-3tau was reported to inhibit PKCµ kinase activity both in vitro and in intact T-cells. In contradiction to these studies, Isobe et al.  showed that a member of the 14-3-3 family, termed ‘Exo1’, activates PKC by two-fold. This finding raised the possibility that the ability of Exo1 to reactivate Ca2+-dependent exocytosis might be based on its stimulatory effect on PKC activity. In the same line, Tanji et al.  also identified 14-3-3 isoforms as activators of PKC in bovine forebrain. Unlike previously reported, the activation of PKC by 14-3-3 was found to be independent of phosphatidylserine and Ca2+-ions and, as such, the study provided an alternative mechanism for the activation of PKC that obviates its translocation to membranes. Later, Acs et al.  reported differential activation of various PKC isoforms, especially PKC epsilon, by 14-3-3 zeta protein in humans.
In addition to PKC, 14-3-3 proteins have also been reported to bind and modulate functional activity of other kinases. For instance, Fantl et al.  reported that 14-3-3 isoforms bind to and enhance Ser/Thr kinase activity of Raf-1, a key mediator of mitogenesis and differentiation, thus promoting Raf-1 dependent oocyte maturation. Next, Takihara et al.  reported that overexpression of 14-3-3β stimulates cellular proliferation and oncogenic transformation of mouse derived fibroblast cell line, by interacting with and enhancing Raf-1 activation and resultant augmentation of signaling in Mitogen-Activated Protein Kinase (MAPK) cascade. In the subsequent year, Rothblum-Oviatt et al.  reported that 14-3-3 proteins function as positive regulators of human Wee1, a tyrosine-specific protein kinase that phosphorylates mitotic inducer Cell division control 2 (Cdc2), and induce Wee1 mediated G2 cell cycle delay. Later, Kim et al.  reported that 14-3-3 interacts with and enhance functional activity of Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A), a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. Furthermore, Fischer et al.  demonstrated that 14-3-3 proteins associate with and activate serine/threonine-specific RAF isoforms (A, B and C) in a isoform-specific manner, thus further elucidating complex mechanism of RAF activation. More recently, Yuasa et al.  demonstrated that Death-Associated Protein Kinase 2 (DAPK2; a Ca2+/calmodulin-regulated serine/threonine kinase)-induced apoptosis is negatively regulated by its interaction with 14-3-3 proteins.
In the current study, we report that Pf14-3-3 isoform I interacts with PfCDPK1, a highly expressed protein in the malaria parasite, which plays key role in multitude of essential cellular processes, including parasite invasion and egress during intra-erythrocytic proliferative stages of the parasite. Preliminary structural modeling and MD simulation studies of Pf14-3-3Idimer/pCDPK1 complex indicated that PfCDPK1 binds to the amphipathic groove of Pf14-3-3Idimer in a thermodynamically stable manner. The interaction is largely driven by charged residues present at the interface of the two proteins, and hydrophobic interactions further stabilize the association. Variation of RMSD and Radius of Gyration indicated conformational fluctuations in Pf14-3-3Idimer upon binding to pCDPK1, which may assist in forming a stable complex via induced-fit mechanism [113,114]. The simulation results were confirmed experimentally by combining various biochemical and biophysical approaches. ELISA based PPI analysis indicated that Pf14-3-3I interacts with PfCDPK1 in a phosphorylation dependent manner. The associated binding strength of the interaction was subsequently quantified by SPR, which indicated that Pf14-3-3I has approximately two fold higher affinity towards pCDPK1 than for CDPK1. In a related unpublished study by our group, the phosphorylation dependent interaction of Pf14-3-3I with PfCDPK1 was validated in merozoites, wherein, by pulling down Pf14-3-3I, PfCDPK1 was detected in a Ca2+-dependent manner . Our finding overlap with the conception that phosphorylation of target protein is a prerequisite for its interaction with 14-3-3. SPR sensograms suggested specificity of PfCDPK1 towards Pf14-3-3I cavity(ies) in terms of shape complementarity and chemical functionality. Kinase assay in the presence of r14-3-3I suggested that Pf14-3-3 binding is not required in regulating PfCDPK1 enzymatic activity in vitro. Furthermore, confocal microscopic imaging in mature schizonts and free merozoites indicated that Pf14-3-3I co-localizes well with PfCDPK1, towards the cell periphery.
Earlier reports suggest that synthetic peptides containing phosphorylated 14-3-3 binding motifs can efficiently inhibit the association of 14-3-3 proteins with their interacting partners, by typically binding to their conserved amphipathic grooves. The first report utilizing peptides as 14-3-3 binding high-affinity antagonists came from Wang et al. , who identified unphosphorylated peptide (PHCVPRDLSWLDLEANMCLP), termed R18, by screening phage display libraries. The peptide bound to the amphipathic groove of 14-3-3, without isoform selectivity. R18 efficiently blocked the interaction of 14-3-3 with Raf-1 kinase, thereby effectively abolishing the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1, and virulence factor exoenzymeS (ExoS) of the pathogenic bacterium Pseudomonas aeruginosa [87,88]. To further enhance R18 activity in cells, a dimeric R18 sequence, termed difopein (dimeric fourteen-three-three peptide inhibitor), was used to dissect the physiological role of 14-3-3/client-protein interaction, and served as a basis for targeting pro-survival function of 14-3-3, as a potential anticancer strategy . Thereafter, other peptide-based inhibitors have been developed, such as macrocyclic cross-linked peptide synthesized by Glas et al.  and Cromm et al.  that inhibit the interaction between human 14-3-3 and ExoS. The so-called ESp peptide (420QGLLDALDLAS430) which is a 14-3-3 binding motif of ExoS, was used as a starting point for designing macrocyclic PPI inhibitor with different cross-link architectures. Later, Milroy LG et al. (2015) synthesized Tau peptide hybrids to inhibit 14-3-3/Tau interaction, a potential drug target for the treatment of Alzheimer's disease . The peptides were designed with an extended hydrophobic area at the C-terminus that targeted the highly conserved pocket within the amphipathic groove of 14-3-3.
Based on the literature review and owing to the fact that well characterized 14-3-3 recognition motifs (Mode I, II and III) and other naturally occurring 14-3-3-binding peptides can be utilized as 14-3-3/client-protein PPI antagonists, we utilized two different phosphopeptides: Mode I (ARSHpSYPA or peptide 1) and Mode II (RLYHpSLPA or peptide 2) (pS: phosphoserine) to inhibit Pf14-3-3/PfCDPK1 interaction. These phosphopeptides were identified by Rittinger et al.  from oriented peptide library screening, and are recognized by all mammalian and S. cerevisiae 14-3-3 isotypes. Preliminary structural modeling and molecular dynamics simulation studies of Pf14-3-3Idimer/peptides (1 or 2) complexes indicated that the peptides bind to the amphipathic groove of Pf14-3-3Idimer in a thermodynamically stable manner. The interaction is largely driven by Hydrogen-bonds between phosphoSer of the peptide ligands and amino acid residues constituting the conserved amphipathic groove of the receptor protein, and the association is further stabilized by hydrophobic interactions. The stable complex formation between Pf14-3-3Idimer and phosphopeptides (1 or 2) suggests effectiveness of the peptides as potential inhibitors of Pf14-3-3Idimer mediated interactions. The simulation results were experimentally confirmed by ITC with r14-3-3I and peptides synthesized de novo. Peptide 1 showed higher binding affinity for r14-3-3I than peptide 2. Much stronger ΔH value for peptide 1 indicates its ability to form a strong network of Hydrogen-bonds and Van Der Waals interactions with Pf14-3-3I protein. The associated unfavorable entropy may be the result of loss of conformational freedom upon Pf14-3-3I/peptide 1 complex formation. In case of peptide 2, interactions are strongly dominated by favorable entropy, with a slight contribution from enthalpy. Positive entropy suggests important contribution from solvation entropy, which results from the loss of water molecules from the binding surfaces of interacting partners when they come close together to form a complex . Small negative ΔH value indicates smaller number of H-bonding interactions contributing to the formation of Pf14-3-3I/peptide 2 complex. Moreover, for peptide 1, binding stoichiometry (N) of 0.3-0.4 indicates binding of one peptide molecule per two or three molecules of r14-3-3I, which may be due to some additional non-specific interactions, apart from specific binding. Interaction with peptide 2 resulted in N ∼ 1.0, which indicates binding of one peptide molecule per molecule of r14-3-3I. Furthermore, both peptides 1 and 2 efficiently blocked the interaction of Pf14-3-3I with PfCDPK1, as confirmed by ELISA and pull-down experiments with recombinantly purified proteins. To dissect the physiological role of Pf14-3-3I/PfCDPK1 interaction, mature P. falciparum schizonts were allowed to rupture and invade into new erythrocytes, in the absence and presence of peptides (1 or 2). Both peptides significantly inhibited the progression of schizonts to ring stage. The mechanistic details of Pf14-3-3I/PfCDPK1 interaction, in context of parasite physiology, has been demonstrated in unpublished data of our group . Since there are no experiments showing that, in vivo, the peptides disrupt only Pf14-3-3I/PfCDPK1 interaction, the percentage of parasite growth defect could partly be due to the abrogation of the interaction of 14-3-3 with its binding partners other than PfCDPK1.
In conclusion, our findings confirm the existence of Pf14-3-3I protein in the malaria parasite P. falciparum, and present insight into its sequence and structural features which may prove to be an initial lead in understanding its function in the parasite. Moreover, we have shown the physiological relevance of Pf14-3-3I/PfCDPK1 interaction in the parasite, as represented schematically in Figure 6. This study would be useful for designing target specific 14-3-3 recognition motif peptides to block the interaction of Pf14-3-3I with its partner protein(s), and for developing a potential antimalarial strategy.
Overall schematic representation.
The authors declare that there are no competing interests associated with the manuscript.
Funding from Science and Engineering Research Board (SERB) (EMR/2016/005644) and Drug and Pharmaceuticals Research Programe (DPRP) (Project No. P/569/2016-1/TDT) for S.S. is acknowledged. S.S. is a recipient of the Innovative Young Biotechnologist Award (IYBA) and National Bioscientist Award from Department of Biotechnology (DBT). R.J. acknowledges University Grant Commission (UGC) for doctoral research fellowship.
R.J. and S.S. designed the experiments. R.J., P.D. and S.G. performed the experiments. S.P. and S.S. contributed reagents. R.J., A.B., M.M., S.S. analyzed the data. R.J., P.D., A.B. and S.S. wrote the manuscript.
Authors acknowledge AutoLab Esprit SPR facility of Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, India and Central Instrumentation Facility (CIF) of SCMM, JNU for flow cytometry. The lab facility of Shiv Nadar University is also acknowledged.
Calcium Dependent Protein Kinase 1
binding affinity constant
Multiple Sequence Alignment
Protein Kinase A catalytic subunit
Protein Kinase A regulatory subunit
Protein Kinase G