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Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

14 Jan 2020-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: This study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target, setting a precedent for the rational design of 14- 3-3 based PPI inhibitors by utilizing 14-2-3 recognition motif peptides, as a potential antimalarial strategy.
Abstract: 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 of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, 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: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, 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.

Summary (4 min read)

Introduction

  • Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps.
  • Overall, this study characterizes 14-3- 3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target.
  • Depending on the biochemical nature of their phosphorylated protein targets, physical association with 14-3-3 proteins can have different functional consequences, resulting in modulation of its enzymatic activity, subcellular sequestration, protein stability and/or alteration of protein-protein interactions (9, 19).
  • Further, the authors report that 14-3-3I 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.

Results

  • Sequence analysis and identification of P. falciparum 14-3-3I MultAlin-based sequence alignment of 14- 3-3 isoforms from Homo sapiens and P. falciparum 3D7 demonstrated patterns of conservation and correlation in Pf14-3-3I protein sequence in light of the well-studied orthologs in humans .
  • The comparable Ramachandran plot characteristics and RMSD value confirmed the reliability of the 3D-structural model of Pf14-3-3Idimer to be taken further for docking and simulation studies.
  • Further, western blot analysis of native 14-3-3I protein in the parasite lysate, by using in-house generated polyclonal mice sera raised against r14-3-3I, confirmed the existence of 14-3-3I protein in the parasite .
  • Updated optimal consensus 14-3-3 binding motifs constructed from amino acid sequences of these 14-3-3 binding phosphopeptides are shown .
  • To probe the associated molecular interactions that regulate the binding affinity of 14-3-3Idimer with pCDPK1, the authors performed MD simulations.

14-3-3I exhibits divergent binding affinities for CDPK1 and pCDPK1 in vitro

  • For ELISA based 14-3-3/CDPK1 PPI analysis, Poly-L-Lysine coated 96-welled microtitre plate was coated with rCDPK1 (or rpCDPK1), followed by addition of increasing concentrations of the prey protein, r14-3-3I.
  • The interaction analysis was done by using monoclonal antibody against GST protein.
  • 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 & binding affinities at RT.
  • IFA was performed on synchronized P. falciparum 3D7 culture to check for colocalization 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, and images were acquired using Nikon A1-R Interaction of P. falciparum 14-3-3I and CDPK1 confocal microscope using the NIS Elements software.

MD Simulation reveals stable complex formation of 14-3-3Idimer with Phosphopeptides 1, 2

  • The peptide 1 was found to bind in the amphipathic groove of the receptor protein, 14-3-3Idimer.
  • The observation was supported by the higher number of intermolecular Hydrogen-bonds that are formed between 14-3-3Idimer and peptide 2 as compared to peptide 1.
  • This was achieved by determining kinetic parameters of the interaction, like binding affinity constant (Ka) and change in enthalpy (ΔH), under sustained salt (150 mM NaCl) and pH (7.4) conditions, at RT.
  • In case of peptide 2, as further injections continued, decline in exothermic heat resulted in sigmoidal curve ending near zero baseline.
  • In Figure 3B, solid line shows the best fit of non-linear experimental data, and the model reproduces experimental data fairly well.

3I protein was enthalpically favorable (ΔH = −11.34 ± 4.27 kcal/mol), whereas entropically

  • Immunoblotting with HRP-conjugated anti-His antibody indicated that phosphorylation status of CDPK1 dictates its interaction with 14-3-3I, as confirmed by ELISA .
  • The same blot stripped and re-probed with HRP-conjugated antiGST antibody served as experimental control to check equal coupling of r14-3-3I (or GST) with Glutathione Sepharose beads in all binding reactions.
  • Towards this, mature schizonts were allowed to invade into erythrocytes in the presence of 12.5 μM concentration of the peptides.
  • Untreated parasites served as control and percentage parasite growth inhibition was measured by flow cytometry, as described in experimental procedures section.

Discussion

  • 14-3-3 is a novel class of dimeric, conserved scaffold proteins that recognize phosphor-serine/threonine (pS/pT) containing conserved binding motifs in a variety of signaling proteins, thus regulating their physiological functions.
  • The epsilon group of 14-3-3 proteins is considered as ancestral and destined to fulfill fundamental cellular functions.
  • SPR analysis indicated that 14-3-3I has approximately two fold higher affinity towards phosphorylated CDPK1, with binding affinity constants (KD) of 1.35 µM & 0.67 µM for 14-3-3I/CDPK1 and 14-3-3I/pCDPK1 interaction, respectively .
  • The peptide exhibited high affinity for different isoforms of 14-3-3 by binding to the amphipathic groove of 14-3-3 via polar and hydrophobic interactions.
  • In conclusion, their 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 of its function in the parasite.

Culture of P. falciparum

  • Cryopreserved P. falciparum parasites (3D7 laboratory strain) were thawed and cultured according to the protocol as described by W. Trager & JB. Jensen (1976) (51).
  • Briefly, parasite cultures were maintained in O + erythrocytes at 2% hematocrit level, in RPMI 1640 medium (Gibco®, USA) supplemented with 0.5% AlbuMAX TM I (Gibco®, USA), 50 mg/L hypoxanthine, 10 mg/L gentamycin (Gibco®, USA) and 2 gm/L sodium bicarbonate.
  • To identify experimentally validated 14-3- 3 interacting partners from prokaryotes & eukaryotes, and collate details of phosphoSer/Thr sites on the target proteins that have been reported to bind directly to 14-3-3 proteins, literature survey followed by mining of publically available databases was done.
  • 18S rRNA was also amplified from genomic DNA (gDNA) of the parasite, as positive control.
  • DNA fragment encoding full-length Pf14-3-3I protein was amplified with the following primer sets: Pf14-3- 3I_Fwd, 5′- TGCGGGATCCATGGCAACATCTGAAGAAT.

Scientific ™) at 4 ºC for 15 minutes. Bacterial

  • Pellet, thus obtained, was resuspended in cell lysis buffer (buffer A) containing 10 mM HEPESNaOH, pH 7.4; 1 mM Ethylene Diamine Tetraacetic Acid (EDTA); 150 mM NaCl; 25μg/ml lysozyme; 3 mM β-MercaptoEthanol (βME), Protease Inhibitor Cocktail (PIC, Roche) and 1mM PhenylMethylSulfonyl Fluoride (PMSF), followed by sonication for 15 minutes with successive pulses of 6 seconds ON and 10 seconds OFF.
  • The formulation was made by thoroughly mixing equal volumes of Freund‟s complete adjuvant and saline containing r14-3-3I protein.
  • Once the anti-serum was raised, native Pf14-3-3I protein was detected in schizonts lysate by immunoblotting.
  • Protein purification was done in a similar manner as the purification of r14-3-3I, except for the following changes in buffers‟ compositions.
  • The slides were washed and mounted with ProLong Gold antifade reagent with DAPI (4',6- diamidino-2phenylindole) and images were acquired using Nikon A1-R confocal microscope using the NIS Elements software.

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 14-3- 3Idimer and CDPK1 from P. falciparum strain 3D7.
  • 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 14-3-3I and CDPK1 as query sequences, against Protein Data Bank (PDB) database (http://www.rcsb.org/) (76, 77).
  • Reliability of the refined structural models of Pf14-3-3Idimer and PfCDPK1 was assessed by examining backbone dihedral angles: phi (Ø) and psi (Ψ) of the amino acid residues lying in the energetically favourable regions of Ramachandran space (83).
  • This was done by using online available tool, PROCHECK v.3.5 (https://www.ebi.ac.uk/thorntonsrv/software/PROCHECK/) (84).

ELISA to confirm 14-3-3I/CDPK1 interaction

  • In all experiments, minimally & maximally phosphorylated CDPK1 protein is represented as rCDPK1 and rpCDPK1, respectively.
  • Surface Plasmon Resonance analysis of binding affinity between CDPK1 and immobilized 14-3-3I rCDPK1 protein was auto-phosphorylated in an in vitro kinase reaction, as mentioned above.
  • Briefly, 5 μM of recombinant 14-3-3I protein 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.
  • Data were fit to the two-state conformational change model by using AutoLab SPR Kinetic Evaluation software provided with the instrument.
  • At least three independent experiments were performed.

Isothermal Titration Calorimetric analysis

  • To calculate kinetic parameters such as binding affinity constant (Ka) for interaction of 14- 3-3I with consensus 14-3-3 binding peptides 1 and 2, Isothermal Titration Calorimetry (ITC) experiments were performed by using MicroCal iTC200 (Malvern Instruments Ltd, UK; at School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India).
  • For this purpose, Interaction of P. falciparum 14-3-3I and CDPK1 recombinant 14-3-3I protein was dialyzed extensively against HEPES-NaCl buffer (10 mM HEPES-NaOH, pH 7.4 and 150 mM NaCl) using Amicon TM Ultra-15 Centrifugal Filter Unit (10 kDa cutoff) before its subsequent use in ITC.
  • Amount of heat produced per injection (corrected data) was analyzed by integration of area under individual peaks by MicroCal ORIGIN 7 software provided by the instrument manufacturer.
  • Experimental data was presented as the amount of heat produced per second (μcal/sec; corrected for heat of dilution of the ligand) following each injection of the ligand into the protein solution, as a function of time .
  • After progression of the parasites to ring stage, erythrocytes were washed with 1X PBS and stained with Ethidium Bromide (EtBr, 10 μM) for 20 minutes at RT, in dark.

Statistical analysis

  • 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.
  • Interaction of P. falciparum 14-3-3I and CDPK1 Acknowledgements: 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.
  • The authors declare that they have no conflicts of interest with the contents of this article.

FOOTNOTES

  • This work has been funded by DST-EMR from the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India.
  • E) Localization of 14-3-3. Anti-r14-3-3I mice serum was used to probe localization of the protein in mature schizonts and free merozoites by using confocal microscopy.
  • The experiment was done twice in triplicates.
  • RMSD: Root Mean Square Deviation; pS: Phosphorylated serine; KD: Affinity constant; DIC: Differential Interference Contrast image.

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Interaction of P. falciparum 14-3-3I and CDPK1
1
Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite
Ravi Jain
1
, Pinki Dey
2
, Sakshi Gupta
3
, Soumya Pati
1
, Arnab Bhattacherjee
2
, Manoj Munde
3
and
Shailja Singh
1,3
*
From the
1
Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Gautam
Buddha Nagar, UP: 201314, India;
2
School of Computational and Integrative Sciences, Jawaharlal Nehru
University, Delhi: 110067, India;
3
School of Physical Sciences, Jawaharlal Nehru University, Delhi:
110067, India;
3
Special Center for Molecular Medicine, Jawaharlal Nehru University, Delhi: 110067,
India.
Running Title: Interaction of P. falciparum 14-3-3I and CDPK1
* To whom correspondence should be addressed: Dr. Shailja Singh:
Special Center for Molecular
Medicine, Jawaharlal Nehru University, Delhi: 110067, India; shailjasngh@gmail.com; Tel.: 011-
26743038; Fax: 011-26742580.
Keywords: Plasmodium falciparum, 14-3-3, 14-3-3I/CDPK1 interaction, CDPK1 mimetic peptides,
parasite growth.
ABSTRACT
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 of target proteins and confer
conformational changes leading to modulation of
their functional parameters. Modulation in
functional activity of kinases has been attributed to
their interaction with 14-3-3 proteins. Herein, we
have characterized Plasmodium falciparum 14-3-3
and its interaction with key kinase of the parasite,
Calcium-Dependent Protein Kinase 1 (CDPK1) by
performing various analytical biochemistry and
biophysical assays. Towards this, we annotated
PF3D7_0818200 as 14-3-3 isoform I through
extensive phylogenetic and comparative sequence
analysis. Molecular dynamics simulation studies
indicated that phosphoSer
64
present in CDPK1
polypeptide sequence (
61
KLGpS
64
) behaves as
canonical Mode I-type (RXXpS/pT) consensus 14-
3-3 binding motif, mediating the interaction. The
protein-protein interaction was validated in vitro
with ELISA and SPR, which confirmed that
CDPK1 interacts with 14-3-3I in a
phosphorylation dependent manner, 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:
ARSHpSYPA and RLYHpSLPA as CDPK1
mimetics, by simulation studies and ITC. Further,
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 (1).
Reversible phosphorylation of serine, threonine or
tyrosine residue has been the most extensively
studied PTM (2). However, very often,
phosphorylation of a protein is not solitary
responsible to modulate its function. Rather,
protein phosphorylation ensures interaction with
its downstream protein-interacting partners which
ultimately regulates its function. 14-3-3 proteins
serve as prototype for such novel class of scaffold
modules that recognize phosphor-serine/threonine
(pS/pT) containing conserved binding motifs in a
variety of signaling proteins. 14-3-3s are highly
evolutionarily conserved dimeric (homo- and
heterodimers), acidic proteins, widespread in
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted January 21, 2020. ; https://doi.org/10.1101/2020.01.14.906479doi: bioRxiv preprint

Interaction of P. falciparum 14-3-3I and CDPK1
2
almost all eukaryotic organisms (35). Although
high degree of sequence conservation among 14-3-
3 isoforms suggests a functional redundancy, the
presence of phenotypes for single and multiple 14-
3-3 knock-out mutants, and differential subcellular
localization of 14-3-3s within a cell suggest that
14-3-3 isoforms selectively bind to their individual
protein ligands with different affinities owing to
the spatiotemporal regulation of the expression of
different isoforms (610). Moreover, the 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
cognate protein partners through canonical
phosphorylated motifs, categorized as: Mode I
(RXXpS/pT), Mode II (RXXXpS/pT) and Mode
III (RXXpS/pTX
1-2
C‟), where X is any amino acid
and pS/pT represents phosphoserine or
phosphothreonine (1116). Mechanistically, 14-3-
3 dimer interacts with its target protein(s) via two
amphipathic grooves harbored by each monomer,
and confers a conformational change that results in
modulating functional parameters of its target
protein(s) (17, 18). Depending on the biochemical
nature of their phosphorylated protein targets,
physical association with 14-3-3 proteins can have
different functional consequences, resulting in
modulation of its enzymatic activity, subcellular
sequestration, protein stability and/or alteration of
protein-protein interactions (9, 19). Earlier reports
also suggest that, 14-3-3 possess chaperone-like
activity akin to that of sHsps (small Heat shock
proteins) and plays a critical role in formation of
14-3-3 mediated aggresomal targeting complex in
response to accumulation of various misfolded
proteins under conditions of cellular stress (20).
Modulation in functional activity of kinases has
also been attributed to 14-3-3 proteins, which have
been described as inhibitors or activators of
calcium and phospholipid dependent Protein
Kinase C (PKC), and an activator of Raf-1 (21
24).
14-3-3 proteins, thus, operate as
multipurpose conformation (allosteric) clamps that
are recruited to hold its phosphorylated cognate
protein(s) in place in response to cellular signaling
pathways, culminating in regulation of 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, 25
28). In this regard, stability of 14-3-3 has been put
forward as a basis for “molecular anvil
hypothesis” according to which the rigid 14-3-3
dimer can induce structural rearrangements in its
partner protein molecule(s), thereby regulating its
functional properties while itself undergoing only
minimal structural alterations (29). Studies on 14-
3-3/client-protein interactions, by utilizing various
biochemical and biophysical tools, may therefore
provide tremendous opportunities for therapeutic
interventions under various 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 PlasmoDB Plasmodium Genomic
Resource database (release 46; updated on 6th
Nov., 2019). The findings of the present study
confirm the presence of 14-3-3 protein in the
malaria parasite. Further, we report that 14-3-3I
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 14-3-3I/CDPK1 protein-protein interaction
(PPI) by utilizing well characterized 14-3-3
recognition motifs as CDPK1 mimetic 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.
Results
Sequence analysis and identification of P.
falciparum 14-3-3I
MultAlin-based sequence alignment of 14-
3-3 isoforms from Homo sapiens and P.
falciparum 3D7 demonstrated patterns of
conservation and correlation in Pf14-3-3I protein
sequence in light of the well-studied orthologs in
humans (Figure 1A). Residues with high
consensus value (>90%) are shaded in red and
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted January 21, 2020. ; https://doi.org/10.1101/2020.01.14.906479doi: bioRxiv preprint

Interaction of P. falciparum 14-3-3I and CDPK1
3
residues with low consensus value (>50%, <90%)
are shaded in blue. α-helices and Nuclear Export
Signal (NES) are also indicated. Five highly
conserved sequence blocks, as identified by Wang
W. and Shakes DC. (1996) were observed, as
shown boxed and shaded red (30). 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 seems to be the
most divergent form of 14-3-3 proteins. 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 identified and highlighted in the overall
Pf14-3-3I architecture (Figure 1B).
X-Ray diffraction based structural model
of Hs14-3-3 epsilon
dimer
was used as a template to
generate three-dimensional coordinates of Pf14-3-
3I
dimer
. After optimal rigid-body superimposition
of Hs14-3-3 epsilon
dimer
with the generated
structural model of Pf14-3-3I
dimer
, overall Root-
Mean-Square Deviation (RMSD) value of the C-
alpha atomic coordinates was found to be 0.63 Å,
suggesting a reliable 3D structure of Pf14-3-3I
dimer
.
Structural model of Pf14-3-3I
dimer
revealed strong
resemblance with it‟s counterparts in 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 on target
proteins. Additionally, both monomers of Pf14-3-
3I
dimer
were found to be oriented in opposite
direction with respect to each other. Helical
regions in one of the monomers are marked from
H1 to H9 (Supporting figure S1A). Assessment of
stereochemical quality and accuracy of the
generated 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 (Supporting
figure S1B). Since, protein structure with ≥90% of
its amino acid residues lying in the most favoured
regions of Ramachandran plot is considered to be
as accurate as a crystal structure at -resolution,
this indicated that the backbone dihedral angles:
phi and psi of the generated Pf14-3-3I
dimer
model
were reasonably accurate. The comparable
Ramachandran plot characteristics and RMSD
value confirmed the reliability of the 3D-structural
model of Pf14-3-3I
dimer
to be taken further for
docking and simulation studies.
Unrooted phylogenetic relationship of
Pf14-3-3 isoforms with their orthologs present
across three major kingdom 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
(Supporting figure S2A). Branches with green, red
and blue squares belong to kingdom plantae,
animalia and fungi, respectively. Detailed
evolutionary relationship of Pf14-3-3 isoforms
with their orthologs from plant non-epsilon group
is shown (Supporting figure S2B).
Transcripts encoding for 14-3-3I protein
was amplified from cDNA prepared from
merozoites, by using pf14-3-3I specific primers, as
mentioned in experimental procedures section.
Detection of desired DNA fragment of 341 bp
confirmed the existence of 14-3-3I encoding
cDNA in the parasite (Figure 1C). Amplification
of transcripts encoding for 18S rRNA (120 bp) by
using pf18s specific primers was taken as positive
control. Further, western blot analysis of native
14-3-3I protein in the parasite (schizonts) lysate,
by using in-house generated polyclonal mice sera
raised against r14-3-3I, confirmed the existence of
14-3-3I protein in the parasite (Figure 1D).
Desired protein band of around 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.
To update optimal 14-3-3 binding
consensus motifs, literature search followed by
mining of publically available databases was done
to identify all experimentally validated 14-3-3
interacting partners present in prokaryotes &
eukaryotes, and collate details of phosphoSer/Thr
target sites on 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 phosphoSer/Thr sites
(Supporting table S1). Updated optimal consensus
14-3-3 binding motifs constructed from amino
acid sequences of these 14-3-3 binding
phosphopeptides are shown (Supporting Figure
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted January 21, 2020. ; https://doi.org/10.1101/2020.01.14.906479doi: bioRxiv preprint

Interaction of P. falciparum 14-3-3I and CDPK1
4
S3A). Further, protein kinases were filtered out as
putative binding partners of Pf14-3-3I by
combining publicly available global phospho-
proteomic datasets of peptides enriched from
schizont stage of P. falciparum, with 14-3-3
binding sequence motifs search (Supporting
Figure S3B). Our results speculate that Pf14-3-3
binds to and regulates the physiological activity of
master kinases of the parasite: CDPK1, Protein
Kinase G (PKG), Protein Kinase A regulatory
subunit (PKA
R
) and Protein Kinase A catalytic
subunit (PKA
C
). Amino acid sequences of
probable 14-3-3 binding phosphopeptides of
CDPK1 are also shown (Supporting Figure S3A).
Molecular Dynamics (MD) Simulation reveals
stable complex formation of 14-3-3I
dimer
with
CDPK1
To probe the associated molecular
interactions that regulate the binding affinity of
14-3-3I
dimer
with pCDPK1, we performed MD
simulations. A schematic representation of the
interactions between 14-3-3I
dimer
and pCDPK1 is
shown in Figure 2A. The phosphorylated serine,
pS64 of pCDPK1 was found to play a key role in
mediating its interaction with 14-3-3I
dimer,
where a
Hydrogen-bond was formed between pS64 of
pCDPK1 and K227 of 14-3-3I
dimer
with a bond
length of 2.92Å. In addition to Hydrogen-bonding,
the 14-3-3I
dimer
-pCDPK1 binding was further
stabilized by hydrophobic interactions between
pS64 of pCDPK1 and Y226, Y555, E557, I597 of
14-3-3I
dimer
located within the amphipathic binding
pocket of the protein. Further comprehensive
analysis of the MD trajectories, i.e., Root Mean
Square Deviation (RMSD), Radius of Gyration
and variation in the Hydrogen-bond formation
between 14-3-3I
dimer
and pCDPK1 were also
reported as a function of simulation time (Figure
2A). The variation of RMSD and Radius of
Gyration in 14-3-3I
dimer
and pCDPK1 clearly
indicated a lesser stability and compactness in the
former compared to pCDPK1. The higher
conformational fluctuations in 14-3-3I
dimer
may
assist in forming a stable complex with pCDPK1
via induced-fit mechanism (31, 32). The higher
stability of the protein-protein complex was also
confirmed by the extensive Hydrogen-bond
formation seen between pCDPK1 and 14-3-3I
dimer
at different time intervals throughout the
simulation and a negative binding energy (-36.8 ±
1.0 kcal/mol) as obtained from MM/GBSA
method. Movie S1 shows the interactions between
14-3-3
dimer
(blue) and pCDPK1 (grey).
14-3-3I exhibits divergent binding affinities for
CDPK1 and pCDPK1 in vitro
For ELISA based 14-3-3/CDPK1 PPI
analysis, Poly-L-Lysine coated 96-welled
microtitre plate was coated with rCDPK1 (or
rpCDPK1), followed by addition of increasing
concentrations of the prey protein, r14-3-3I. The
interaction analysis was done by using monoclonal
antibody against GST protein. A concentration
dependent binding between rCDPK1 (or
rpCDPK1) and the prey protein r14-3-3I was
observed (Figure 2B). PPI analysis by ELISA
suggested that phosphorylation status of CDPK1
dictates its interaction with 14-3-3I. To quantitate
the interaction between rCDPK1 (or rpCDPK1)
and r14-3-3I, SPR analysis was performed by
utilizing AutoLab Esprit SPR. r14-3-3I was
immobilized at an average density of 4.3 ng per 1
mm
2
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 & 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.
The concentration dependent real-time
sensorgrams along with K
D
values of interactions
are shown in figure 2B. rCDPK1 and rpCDPK1
showed differential binding affinities r14-3-3I,
with K
D
values varying from 0.67 ± 0.0036 μM
(14-3-3I/pCDPK1) and 1.35 ± 0.0083 μM (14-3-
3I/CDPK1). Also, SPR sensograms suggested
specificity of CDPK1 towards 14-3-3I cavity(ies)
in terms of shape complementarity and chemical
functionality.
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,
and images were acquired using Nikon A1-R
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted January 21, 2020. ; https://doi.org/10.1101/2020.01.14.906479doi: bioRxiv preprint

Interaction of P. falciparum 14-3-3I and CDPK1
5
confocal microscope using the NIS Elements
software. 14-3-3I protein was found to co-localize
very nicely with CDPK1 protein towards cell
periphery in mature schizonts and free merozoites.
MD Simulation reveals stable complex formation
of 14-3-3I
dimer
with Phosphopeptides 1, 2
In Figure 3A, the interactions of 14-3-
3I
dimer
with phosphopeptides 1 and 2 were studied
by performing 20 ns MD simulations. The peptide
1 was found to bind in the amphipathic groove of
the receptor protein, 14-3-3I
dimer
. Here, three
hydrogen bonds were formed between the
phosphorylated Serine in peptide 1 and K306,
K379, S302 of the receptor protein, in addition to
hydrophobic interactions that further stabilized the
14-3-3I
dimer
-peptide complex. Similarly, the
phosphorylated Serine of peptide 2 formed a
Hydrogen-bond with N188 of 14-3-3I
dimer
, along
with stabilizing hydrophobic interactions. A
comparative study using time-dependent variations
in RMSD and Radius of Gyration, however,
revealed that 14-3-3I
dimer
-peptide 2 complex was
more stable and slightly more compact compared
to when 14-3-3I
dimer
interacts with the peptide 1.
The observation was supported by the higher
number of intermolecular Hydrogen-bonds that are
formed between 14-3-3I
dimer
and peptide 2 as
compared to peptide 1. This also accounts for the
higher negative binding energy observed for 14-3-
3I
dimer
-peptide 2 complex (-41.8 ± 1.0 kcal/mol)
relative to 14-3-3I
dimer
-peptide 1 complex (-67.2 ±
0.1 kcal/mol). Movies S2 and S3 show the
interactions of 14-3-3
dimer
(blue) with peptides 1 &
2 (yellow), respectively.
Phosphopeptides 1 & 2 interact with 14-3-3I in
vitro
After affirming 14-3-3I
dimer
interaction
with phosphopeptides 1 & 2 through molecular
dynamics simulation studies, we further sought to
establish the knowledge of binding modes of
complexation between r14-3-3I and the peptides,
by employing ITC. This was achieved by
determining kinetic parameters of the interaction,
like binding affinity constant (K
a
) and change in
enthalpy H), under sustained salt (150 mM
NaCl) and pH (7.4) conditions, at RT. ΔH along
with K
a
values were then utilized for calculation of
additional parameters, like binding free energy
G, equivalent to -RT.lnK
a
) and entropy S,
equivalent to H ΔG)/T]. The representative
binding isotherms resulting from titration of
peptide 1 or 2 with r14-3-3I are represented in
figure 3B. Binding isotherm with peptide 1 was
monophasic in nature, reaching a plateau phase
indicating saturation of r14-3-3I binding sites. In
case of peptide 2, as further injections continued,
decline in exothermic heat resulted in sigmoidal
curve ending near zero baseline. On the basis of
the nature of the curves, the data were fitted by
using single-site binding model. In Figure 3B,
solid line shows the best fit of non-linear
experimental data, and the model reproduces
experimental data fairly well. On the basis of K
a
values, binding strength was found to be higher in
case of 14-3-3I/peptide 1 (K
a
: 1.7 X 10
6
± 6.8 X
10
5
M
-1
) than in case of 14-3-3I/peptide 2 (K
a
: 8.3
X 10
5
± 2.67 X 10
5
M
-1
). Moreover, it was
observed that the binding of peptide 1 with r14-3-
3I protein was enthalpically favorable H =
−11.34 ± 4.27 kcal/mol), whereas entropically
unfavourable (TΔS = -2.8 kcal/mol 15-20%),
resulting in strong binding free energy G = -8.5
kcal/mol 15-20%). For 14-3-3I/peptide 2
complex, the interaction was enthalpically as well
as entropically driven H = −1.52 ± 0.096
kcal/mol, S = 6.5 kcal/mol 15-20%) and ΔG
= -8.0 kcal/mol (± 15-20%).
Phosphopeptides 1 & 2 as CDPK1 mimetic to
antagonize 14-3-3I/CDPK1 interaction
ELISA based 14-3-3/CDPK1 PPI analysis
was performed in the presence of varying
concentrations of phosphopeptides 1 & 2. The
interaction analysis was done by using monoclonal
antibody against GST protein. Concentration
dependent inhibition of binding between
rpCDPK1 and r14-3-3I was observed (Figure 4A).
To further confirm 14-3-3I/CDPK1 interaction
inhibition by the peptides, 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
CDPK1 dictates its interaction with 14-3-3I, as
confirmed by ELISA (Figure 4B). Moreover,
inhibition of binding between rCDPK1 (or
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The copyright holder for this preprintthis version posted January 21, 2020. ; https://doi.org/10.1101/2020.01.14.906479doi: bioRxiv preprint

Citations
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Journal ArticleDOI
25 Aug 2020-Mbio
TL;DR: A novel signaling complex that plays a key role in merozoite invasion of RBCs is identified and could serve as a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.
Abstract: Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways Exposure of P falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1) Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P falciparum merozoites A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasitesIMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites

11 citations


Cites background from "Interaction of 14-3-3I and CDPK1 me..."

  • ...binding with Pf14-3-3I and block blood-stage parasite growth (31)....

    [...]

Posted ContentDOI
20 Jan 2020-bioRxiv
TL;DR: A novel signaling complex that plays a key role in merozoite invasion of RBCs is identified providing a novel approach for the development of inhibitors to block the growth of blood stage malaria parasites.
Abstract: Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/ low K+) out of which 143 were Ca2+-dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using co-immunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multi-protein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+ dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multi-protein complex. Disruption of the multi-protein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood stage growth of malaria parasites. ImportanceInvasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we have used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation dependent multi-protein complex including signaling kinases assembles during the process of invasion. Disruption of this multi-protein complex impairs merozoite invasion of RBCs providing a novel approach for the development of inhibitors to block the growth of blood stage malaria parasites.

3 citations

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"Interaction of 14-3-3I and CDPK1 me..." refers methods in this paper

  • ...We used a non-bonded cutoff distance of 8Å, and Particle Mesh Ewald (PME) algorithm handled the long ranged electrostatic interactions (93)....

    [...]

Frequently Asked Questions (19)
Q1. What are the contributions in "Interaction of 14-3-3i and cdpk1 mediates the growth of human malaria parasite" ?

Herein, the authors have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 ( CDPK1 ) by performing various analytical biochemistry and biophysical assays. Overall, this study characterizes 14-33I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. Reversible phosphorylation of serine, threonine or tyrosine residue has been the most extensively studied PTM ( 2 ). CC-BY-ND 4. 0 International license available under a ( which was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. 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. 

R18 efficiently blocked the binding of 14-3-3 to Raf-1 kinase, a physiological ligand of 14-3-3, thereby effectively abolishing the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1, and virulence factor exoenzyme S (ExoS) of the pathogenic bacterium Pseudomonas aeruginosa (44). 

the structural changes conferred in 14-3-3Idimer upon the binding of CDPK1 might assist a stable complex formation between them. 

The phosphorylated serine, pS64 of pCDPK1 was found to play a key role in mediating its interaction with 14-3-3Idimer, where a Hydrogen-bond was formed between pS64 of pCDPK1 and K227 of 14-3-3Idimer with a bond length of 2.92Å. 

The peptide exhibited high affinity for different isoforms of 14-3-3 by binding to the amphipathic groove of 14-3-3 via polar and hydrophobic interactions. 

Phosphopeptides 1 & 2 interact with 14-3-3I in vitroAfter affirming 14-3-3Idimer interactionwith phosphopeptides 1 & 2 through molecular dynamics simulation studies, the authors further sought to establish the knowledge of binding modes of complexation between r14-3-3I and the peptides, by employing ITC. 

inhibition of binding between rCDPK1 (orInteraction of P. falciparum 14-3-3I and CDPK1rpCDPK1) and r14-3-3I was readily observed in the presence of peptides 1 & 2. 

In the concurrent year, Wendy J. Fanti et al (1994) reported that 14-3-3 isoforms bind and enhance serine/threonine kinase activity of Raf-1, a key mediator of mitogenesis and differentiation, thus promoting Raf-1 dependent oocyte maturation (24). 

Earlier reports suggest that syntheticpeptides 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 the conserved amphipathic groove of 14-3-3s. 

Residues involved in dimerization (solid circles) and phosphopeptide binding (solid squares) are conserved in all 14-3-3 Isotypes, except Pf14-3-3II which appears to be the most divergent form. 

the authors have shown the interaction of Pf14-3-3I with CDPK1, an important kinase of the parasite involved in motility and apical organelle discharge critical for invasion process. 

SPR analysis indicated that 14-3-3I has approximately two fold higher affinity towards phosphorylated CDPK1, with binding affinity constants (KD) of 1.35 µM & 0.67 µM for 14-3-3I/CDPK1 and 14-3-3I/pCDPK1 interaction, respectively (Figure 2C). 

In a related study by their group, the phosphorylation dependent interaction of 14-3-3I with CDPK1 was validated in merozoites, wherein, by pulling down 14-3-3I, CDPK1 was detected in a Ca 2+ dependent manner (42). 

Experimental data was presented as the amount of heat produced per second (μcal/sec; corrected for heat of dilution of the ligand) following each injection of the ligand into the protein solution, as a function of time (minutes). 

Immunoblotting with HRP-conjugated anti-His antibody indicated that phosphorylation status of CDPK1 dictates its interaction with 14-3-3I, as confirmed by ELISA (Figure 4B). 

Untreated parasites served as control and percentage parasite growth inhibition was measured by flow cytometry, as described in experimental procedures section. 

14-3-3I protein was found to co-localize very nicely with CDPK1 protein towards cell periphery in mature schizonts and free merozoites. 

their phylogenetic analysis shows that despite having evolved separately since the early eukaryotes, Pf14-3-3I protein has followed convergent evolution with plant nonepsilon group, as depicted in the phylogenetic model (Figure S2). 

This study would be useful for designing target specific 14-3-3 recognition motif peptides to block interaction of Pf14-3-3I with its cognate proteins in the parasite, and develop as potential antimalarial strategy.