Cross-Talk between Malarial Cysteine Proteases and
Falstatin: The BC Loop as a Hot-Spot Target
Srinivasan Sundararaj1, Ajay K. Saxena2, Ruby Sharma2, Kapil Vashisht1, Supriya Sharma1,
Anup Anvikar1, Rajnikant Dixit1, Philip J. Rosenthal3, Kailash C. Pandey1*
1 Host–Parasite Interaction Biology Group, National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India, 2 Structural Biology
Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 3 Department of Medicine, San Francisco General Hospital, University of California San
Francisco, San Francisco, California, United States of America
Abstract
Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and
Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined
functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are
regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of
Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial
for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes
and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our
study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis,
hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops,
inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of
falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the
development of anti-malarial agents based on protease-inhibitor interactions.
Citation: Sundararaj S, Saxena AK, Sharma R, Vashisht K, Sharma S, et al. (2014) Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a
Hot-Spot Target. PLoS ONE 9(4): e93008. doi:10.1371/journal.pone.0093008
Editor: Matthew Bogyo, Stanford University, United States of America
Received October 28, 2013; Accepted February 27, 2014; Published April 3, 2014
Copyright: ß 2014 Sundararaj et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work in the laboratory is supported by Department of Biotechnology, Government of India (BT/HRD/35/02/2006, dated 05.02.2010). KCP is a recipient
of a Ramalingaswami Fellowship. The authors thank NIMR for extramural funds for setting a new laboratory. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kailashcp@mrcindia.org
bridges and hydrophobic interactions between the prodomain and
the mature domain play crucial roles [10].
Since many cysteine proteases have broad specificity, it is
important to regulate their activity. Inhibitors of cysteine proteases
(ICPs) of malaria parasites were discovered based on their
similarity to the Trypanosoma cruzi ICP chagasin [11,12]. P.
falciparum expresses the ICP falstatin, which appears to facilitate
the invasion of erythrocytes by asexual merozoites by inhibiting
host and/or parasite cysteine proteases [13]. Similarly, PbICP, the
falstatin homologue in P. berghei, appears to facilitate hepatocyte
invasion by sporozoites and to block programmed cell death by
hepatocytes infected with liver stage parasites [14]. PyICP, the
homologue from P. yoelii, appears to play a similar role in
hepatocytes [14]. Falstatin is a competitive and reversible inhibitor
of falcipains [13]. It has previously been shown by structural
analysis of chagasin–FP2 and PbICP-FP2 complexes that three
ICP loops, known as BC (also named L2), DE (L4) and FG (L6),
bind to the target proteases [12,15]. The C-terminal inhibitory
domain of PbICP binds with FP2 in a 1:1 complex, and the solved
structure suggests that PbICP is a member of the I42 class of
chagasin-like protease inhibitors.
Structural studies have helped to explain the mechanism of
inhibition of falcipains by small molecule inhibitors [16,17]. For
ICPs, the structures of chagasin–FP2 and PbICP-FP2 demon-
Introduction
Plasmodium falciparum and P. vivax are the most predominant
human malaria parasites worldwide. According to the latest WHO
estimate, 219 million cases of malaria and an estimated 660,000
deaths from malaria occurred in 2010 [1]. Control measures for
malaria are seriously hindered by resistance of malaria parasites to
many available anti-malarial drugs [2]. Resistance against
artemisinins, the most important new class of effective drugs, is
also emerging [3]. Therefore, new anti-malarial drugs, particularly
acting against new biochemical targets, are needed. Among
potential new targets for anti-malarial chemotherapy are Plasmodium proteases. Proteases are druggable targets; at present protease
inhibitors are licensed and in clinical development to treat multiple
diseases, including osteoporosis, diabetes, cancer, hypertension
and viral infections.
Among falcipain family cysteine proteases, key enzymes in
erythrocytic parasites appear to be falcipain-2 (FP2) and falcipain3 (FP3), which are major hemoglobinases of P. falciparum, and in P.
vivax, vivapain-2 (VP2), vivapain-3 (VP3) and vivapain-4 (VP4) [4].
Structural and biochemical analysis of falcipains showed that they
have specific domains with defined functions. These include the
trafficking domain, inhibitory domain, refolding domain and
hemoglobin binding domain [5–9]. A recent study also clarified
the mechanism of auto-activation of falcipains, whereby salt
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Interactions between Cysteine Proteases-Falstatin
strated that three loops play important roles in binding to the
target proteases. However, it is not known how specifically falstatin
interacts with cysteine proteases. In this study we investigated the
role in falcipain binding of each loop of falstatin. We found that
only the BC loop appears to plays a central role in protease
inhibition.
Materials and Methods
Ethics Statement
Malaria Parasite Bank of National Institute of Malaria
Research, New Delhi, has been approved by ethical committee
for in vitro culture of P. falciparum.
Z- Leu-Arg-AMC (7-amino-4-methylcoumarin) was from Taurus Scientifics. Wild type and mutant peptides were purchased
from Biochem Life Sciences, India. Restriction endonucleases,
polymerases and T4 ligases were from Fermentas. All other
reagents and chemicals were as mentioned in the text.
Figure 2. Falstatin is a multimer. Native falstatin was characterized
by gel filtration and native PAGE analysis. The elution profile of falstatin
and its migration in native PAGE (right inset) predict a size of ,450 kDa.
Lane A: 660 kDa and 440 kDa markers; Lane B: 43 kDa marker; Lane C:
8 mg of active falstatin; Lane D: 12 mg of active falstatin. The functional
activities of different peaks were analyzed by hemoglobin hydrolysis
observed in 15% SDS-PAGE (left inset). Gel filtration analysis and activity
assay of different peaks were repeated more than two times. The blue
and red lines indicate the absorbance of proteins and conductance of
buffer, respectively.
doi:10.1371/journal.pone.0093008.g002
Cloning of Wild Type and Mutant Falstatin
Amplification of falstatin fragments was done by PCR using P.
falciparum cDNA and an earlier described procedure [13]. The
amplified DNA fragments were purified by gel extraction, ligated
directly into the pGEM-T vector and transformed in Escherichia coli
JM109 competent cells using a Promega TA cloning kit. The wild
type, and mutants (Asn 287, Phe 397) of falstatin were constructed
to study the role of BC and FG loops. The wild type, and mutants
(Asn 287 to Ala 287, Phe 397 to Ala 397) of falstatin were
constructed to study the role of BC (L2) and FG (L6) loops. The
signal sequence was deleted, and expressed the wild type and the
mutants of falstatin as described earlier [13]. All mutants of
falstatin were acquired by overlap extension PCR [18]. Mutant
sequences were confirmed by DNA sequencing. Wild type and
mutant falstatins were amplified from the falstatin-pGEM-T
plasmid, digested with BamHI and Hind III and ligated in a
pQE30 expression vector (Qiagen).
imidazole, 3 mM b-mercaptoethanol, sonicated with a 20 sec
pulse and 1 min gap per cycle for 7 cycles and centrifuged at
12,000 g. The supernatant was then incubated with pre-charged
Ni-NTA resin (Qiagen) for 1 hour, washed with 50 mM imidazole
and eluted with 100–300 mM imidazole, using EKTA Prime Plus
purification system from GE Health Care. The eluted protein was
concentrated using a 10 kDa cut-off membrane (Millipore), loaded
on Sephacryl S-200 HR gel filtration column pre-equlibrated with
50 mM phosphate buffer pH 8, 150 mM NaCl, 5% glycerol and
concentrated to 3 mg/ml. Gel filtration markers (Ferritin,
660 kDa; Aldolase, 440 kDa; ovalbumin, 43 kDa) were from GE
Health Care.
Expression and Purification of Falstatin and its Different
Constructs
Various constructs of falstatin were transformed in E. coli M15
(pREP4) cells (Qiagen) and expressed with 0.5 mM IPTG at 33uC
for 4 hours. The pellets were suspended in 50 mM phosphate
buffer pH 8, 500 mM NaCl, 1 mM phenyl methyl sulfonyl
fluoride (PMSF), 1 mM benzamidine hydrochloride, 10 mM
Figure 1. Alignment of falstatin with other ICPs. A multiple sequence alignment was performed with falstatin and ICPs from Cryptosporidium
parvum (cryptostatin), Trypanosoma cruzi (chagasin), and Plasmodium berghei (PbICP). This alignment predicted four major loop regions; L0, BC (L2),
DE (L4) and FG (L6) in falstatin. The peptides used in this study are underlines, and residues mutated in the described studies are in bold type.
doi:10.1371/journal.pone.0093008.g001
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Interactions between Cysteine Proteases-Falstatin
incubated with the proteases before adding hemoglobin. For all of
these hydrolysis reactions were monitored by measuring absorbance at 410 nm.
To confirm the specificity of the peptide based on the BC loop
(LDSVN287GN289GF291VW), 5 mg trypsin (in 100 mM Tris,
pH 7.4), 5 mg pepsin (in 100 mM acetate buffer, pH 4.0), or 5 mg
plasmepsin 4 (P. vivax aspartic protease in 100 mM acetate buffer,
pH 4.5), [27] were pre-incubated with and without 16 mM of
peptide. After 20 minute incubations, 8 mg of hemoglobin was
added and reaction products after 3 hour incubations were
analyzed using 15% SDS-PAGE.
Modeling of Falstatin-FP2, Falstatin-FP3 and Falstatin-VP2
Complexes
The coordinates of crystal structures of mature domains of FP2
(244–284 aa, PDB-1YVB), [9] and FP3 (8–249 aa, PDB-3BWK),
[16,17] were obtained from the NCBI protein database. The
Phyre server [19] and Modeler V 9.10 [20] were used to model the
structures of VP2 and falstatin.
The structural model of falstatin was obtained using the
PHYRE server, which used the chagasin crystal structure (PDB2OUL), [12] as best input template. We also built the falstatin
model with the Modeler V 9.10 program [20] using the PbICP-C
crystal structure (PDB-3PNR), [14] as input template. The model
was evaluated based on best Z-DOPE score. The initial complexes
of falstatin-FP2, falstatin-FP3 and falstatin-VP2 were obtained
with the COOT program [21] using chagasin-FP2 (PDB-2OUL),
[12] and PbICP-FP2 (PDB-3PNR), [15] as input templates. We
used principal protein-protein docking server CLUSPRO [22] to
obtain a set of possible complexes. The server yielded the best
docking complexes of falstatin-FP2, falstatin-FP3 and falstatinVP2. Energy minimization was performed using the GROMACS
program [23] on all three complexes of falstatin using 200 steps of
steepest descent and 500 steps of conjugate gradient algorithm.
The protein-protein interactions server [24] was used to identify
all interacting residues at complex interface and their solvent
accessibility. The free binding energy between both chains of the
complex was calculated using the DCOMPLEX program [25].
Measurement of Enzyme Activity using a Fluorogenic
Substrate
To assess hydrolysis of a fluorogenic peptide, FP3, VP2 and FP2
(30 nM) were incubated with and without 1 mM E-64 and 50 nM
falstatin before adding 25 mM Z-Leu-Arg-AMC. The wild and
mutant falstatin were incubated in 100 mM sodium acetate,
pH 5.5, 8 mM DTT. For peptide mediated inhibition, a wild type
or mutant peptides (16 mM) were incubated with 30 nM VP2, FP3
and FP2 for 35 min before adding substrate. Fluorescence
(excitation, 355 nm; emission, 460 nm) resulting from hydrolysis
of substrate was continuously measured for 15 min at room
temperature as described earlier [26,28].
Parasite Culture
P. falciparum strain 3D7 was cultured in human erythrocytes
(procured from the Indian Red Cross Society) using 2%
hematocrit in RPMI medium 1640 supplemented with 10%
human serum [29]. Synchronization was maintained by treatment
with 5% D-sorbitol [30]. Synchronized ring-stage parasites were
pre-incubated
with
wild
type
peptide
and
mutant
peptide
(LDSVN287GN289GF291VW)
(LDSVN287GA289GF291VW) based on the BC loop (25.0 mM)
or E-64 (10 mM), and after 30 hours of incubation slides were
prepared, stained with Giemsa and examined for morphological
changes.
Hemoglobin Hydrolysis
To assess the hydrolysis of hemoglobin, 8 mg of hemoglobin was
incubated with 300 nM of FP2, FP3, or VP2 with and without
10 mM E-64 or 500 nM falstatin. Wild type and mutant peptides
at different concentrations (8–32 mM) were incubated with the
above enzymes before incubation with 8 mg of hemoglobin at
37uC for 3 hours. Reaction products were resolved by 15% SDSPAGE and identified by staining with Coomassie as previously
described [26].
To assess hemoglobin hydrolysis by spectrophotometry, proteases as mentioned above were incubated with and without 10 mM
E-64 or 500 nM falstatin before adding 15 mg of hemoglobin. The
above reactions were incubated in 100 mM sodium acetate,
pH 5.5, 8 mM reduced glutathione in 500 ml reaction mixture.
Equal amounts of wild type and mutant peptides (16 mM) were
Hemozoin Assay
Synchronized ring-stage parasites (200 ml with 6% parasitemia)
were treated with two different concentrations of wild type peptide
Figure 3. Models depicting the interactions of wild type peptide and VP2 (A), FP3 (B), and FP2 (C). 3D models depict interactions of the
BC loop peptides (turquoise) with the active site (green) regions of VP2 (A), FP3 (B) and FP2 via hydrogen bonds and hydrophobic interactions.
doi:10.1371/journal.pone.0093008.g003
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Interactions between Cysteine Proteases-Falstatin
Figure 4. Hydrogen bonds are crucial between the BC loop of falstatin and cysteine protease. Hemoglobin and fluorescence peptide
hydrolysis were done in the presence of wild type peptide (LDSVN287GN289GF291VW), mutant peptides (LDSVN287GA289GA291VW and
LDSVN287GA289GF291VW), and mutants of falstatin (Asn 287, Phe 397). The Inhibitory property of wild type peptide and mutant peptides (16 mM)
based on a BC loop were assessed by using fluorescence peptide (Z-Leu-Arg-AMC) with VP2 (Fig. 4A) or FP3 (Fig. 4B).The percentage of fluorescence
units were measured and calculated for the hydrolysis of the fluorogenic substrate (A, B). Similarly, the Inhibitory property of wild type peptide and
mutant peptide were assessed by using spectrophotometer at 410 nm (Fig. 4C). Error bars are shown based on means from two independent assays.
Hemoglobin hydrolysis by VP2 (Fig. 4D) and FP3 (Fig. 4E) were also assessed in the presence of wild type (LDSVN287GN289GF291VW) and mutant
peptide (LDSVN287GA289GA291VW) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE.
doi:10.1371/journal.pone.0093008.g004
(25.0 mM and 50.0 mM). After 30 hours slides were prepared to
confirm the development of early schizonts.
Schizonts were treated with 800 ml of 2.5% SDS in 0.1 M
sodium bicarbonate pH 8.8, and mixed at room temperature for
20 min. The supernatant was removed after centrifugation at
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13,000 rpm for 10 min. The pellet was washed twice with 1ml of
2.5% SDS in 0.1 M sodium bicarbonate (pH 8.8), and finally
resuspended in 500 ml of 5% SDS and 50 mM NaOH. The
quantity of monomeric heme was measured at 405/750 nm as
described earlier [31].
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Interactions between Cysteine Proteases-Falstatin
Figure 5. Only the BC loop appears to plays a central role in protease inhibition. Peptides based on the DE, FG and L0 loops of falstatin
also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE.
As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of P.vivax (PvPM4) under optimal conditions and
hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent
batches of each enzyme.
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Interactions between Cysteine Proteases-Falstatin
Figure 6. Effect of BC peptide on cultured P. falciparum. A Morphology of parasites treated with wild type and mutant BC peptides.
Synchronized ring-stage parasites were incubated with the wild type (LDSVN287GN289GF291VW) and the mutated peptide (LDSVN287GA289GF291VW)
(25.0 mM) or E-64 (10 mM), and after incubation parasites were stained with Giemsa. Morphology of parasites was analyzed with two independent
cultures of P. falciparum. B. Impact of wild type BC peptide on free heme formation. Synchronized ring-stage parasites were incubated
without inhibitor or with the peptide or E-64 at the indicated concentrations, and after 30 hours free heme was quantified by spectroscopy.
Percentages of control free heme are shown based on means from two independent assays.
doi:10.1371/journal.pone.0093008.g006
The BC Loop Plays a Pivotal Role in Cysteine Protease
Inhibition
Results
Similarity between Falstatin and Other ICPs
The structure of PbICP-FP2 suggests that the C-terminus of
PbICP binds to the target protease via its four loops (L0, BC, DE
and FG) [15]. To understand the roles of falstatin loops, mutants
were designed by changing loop amino acids (Asn287 from BC and
Phe397 from FG) to alanine. These point mutants, wild falstatin
(Fig. S1A), and the proteases VP2, FP2 and FP3 were constructed,
expressed and purified (Fig. S1B and Fig. S1C). The inhibitory
activities of wild and mutant falstatins were compared by
measuring their effects on hemoglobin hydrolysis by FP3. Results
with the two mutants showed that the mutated residues are not
crucial for inhibition of FP3 (Fig. S2). Based on these data, we used
molecular modeling of falstatin in complex with VP2, FP3 and
FP2 to predict key inhibitory falcipain domains. Our models
predicted that the sequence corresponding to the BC loop fits into
the active site of VP2 (Fig. 3A), FP3 (Fig. 3B) and FP2 (Fig. 3C).
To validate our in silico results, we studied the effects on
hydrolysis of a peptidyl substrate and hemoglobin in the presence
of a wild type peptide (LDSVN287GN289GF291VW) based on the
BC loop of falstatin. The peptide caused a .75% decrease in
hydrolysis of the fluorogenic substrate, Z-Leu-Arg-AMC (Fig. 4A,
4B). The peptide also inhibited the hydrolysis of the natural
substrate hemoglobin, measured spectrophotometrically, by VP2
(Fig. 4C) or, measured by SDS-PAGE, by VP2 or FP3 (Fig. 4D
and 4E).
In contrast, peptides based on the DE, FG and L0 loops of
falstatin did not inhibit hemoglobin hydrolysis by VP2 (Fig. 5A) or
FP2 (Fig. 5B) or FP3 (Fig. 5C and Fig. 5D and 5E). Although,
there was a difference in inhibitory potency between the wild type
peptide and falstatin, but both were efficient inhibitors of malarial
cysteine proteases. The difference of inhibition between the wild
type and the mutant peptide in two different assays (Fig. 4B and
4E) was due to different level of sensitivity. The hydrolysis of
Structural studies of chagasin and PbICP led to the identification of crucial loops in the ICP family of proteins. Falstatin and its
plasmodial homologues have molecular masses much higher than
those of other ICPs, and so it was unclear if the structural
determinants of inhibition by falstatin are similar to those of non
plasmodial ICPs [11,12]. Our first objective was to identify and
characterize the determinant of inhibition in falstatin. We aligned
the sequences of falstatin and PbICP with those of other ICPs.
Falstatin and it plasmodial homologues have long stretches of
amino acids that are absent in chagasin and homologues from
Leishmania mexicana and Cryptosporidium parvum [11,12,32] (Fig. 1).
Overall, the sequence similarity between falstatin and chagasin is
,20%, and that between falstatin and PbICP is ,38%. Based on
homology with the solved structures of chagasin and PbICP
[12,15], we identified four major loops, labeled as L0, BC (L2), DE
(L4) and FG (L6) (Fig. 1).
Falstatin is a Multimer
Under reducing and denaturing conditions the apparent
molecular mass of falstatin was estimated to be 45 kDa [13].
Based on elution patterns in gel filtration and analysis with native
PAGE (right inset), falstatin appeared to be multimeric, with
approximate size of the multimer of 450 kDa (Fig. 2), indicating a
complex of 10 units of falstatin. The functional activities of
different peaks of gel filtration chromatography suggested that only
a 450 kDa peak is active (left inset). This result contrasts with data
for PbICP and chagasin, which indicate that these inhibitors
function as monomers [12,15] (Fig. 2).
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Interactions between Cysteine Proteases-Falstatin
hemoglobin, by proteases of other classes, the serine protease
trypsin and the aspartic proteases pepsin and P.vivax plasmepsin-4
(PvPM4), [27] was unaffected by the peptide (Fig. 5F). Thus, the
BC loop peptide mimicked wild falstatin to block hemoglobin
hydrolysis by plasmodial cysteine proteases.
function. Multiple sequence analysis identified three falstatin loops
which shared moderate similarity (,40%) with the related ICPs
chagasin and PbICP, and computational analysis suggested key
hydrogen bonds and hydrophobic interactions between falstatin
and falcipains. Specifically, a peptide representing the falstatin BC
loop, but not peptides representing the DE or FG loops, inhibited
FP2, FP3 and VP2, demonstrating the key role of this loop in
mediating falstatin function. Parasite treated with peptide showed
distinct food vacuole abnormalities with undegraded hemoglobin,
further hemozoin assay correlated the dose dependent inhibition
of malarial cysteine proteases, which was further evidenced by the
reduction in hemoglobin degradation. Mutation of residues
predicted to mediate inhibitor-enzyme interactions ablated the
inhibitory function of the peptide. Taken together, these results
demonstrate the key role of the falstatin BC loop in mediating
endogenous inhibition of cysteine proteases by P. falciparum.
The complex of chagasin-FP2 and PbICP-FP2 demonstrated
the importance of BC, DE and FG loops, which bind to the target
proteases. However, using peptides based on these loops of
falstatin suggest that only a BC loop inhibit the malarial cysteine
proteases of P. falciparum and P. vivax. It was further reported in
Leishmania mexicana ICP, DE loop peptide also have no direct role
in inhibition [34]. It may be possible that DE and FG loops are not
require for direct inhibition but may play important role in
stability of falstatin-cysteine protease complex. Nonetheless, the
interaction between the binding loop and the active site are not
conserved in both the endogenous inhibitors. In case of PbICP,
Gly231 of the inhibitor and Trp206 of FP2 form a hydrogen bond
[15], while in case of falstatin, Asn289 of the inhibitor forms three
hydrogen bonds with Asn21 and Cys22 of the active site of VP2,
and one hydrogen bond with Cys41 of the active site of FP3.
Inhibition of a wide range of cysteine proteases by macromolecule
inhibitors like falstatin, PbICP and chagasin, suggest that the BC
loop can adopt more than one alternative conformation for
optimal and efficient inhibition. But neither falstatin nor PbICP is
able to inhibit cathepsin B, because of a loop region that occludes
substrate from binding to the primed subsites of the active site
cleft. The steric repulsion between the two loops (L3 and L4) of
PbICP and the occluding loop of cathepsin B has been reported
which inhibit the interaction between protease and inhibitor [15].
Unlike all other known monomeric endogenous inhibitors,
falstatin is multimeric forms and thus may favor some additional
level of interactions. The close homolog in P. Berghei, PbICP is
active in monomeric state having binding site in a single cavity, but
multimerization of falstatin may cause allosteric modulation of
inhibitory potency. The different structural state of falstatin may
be reason to inhibit distant related cysteine proteases, human
caspase-3, caspase-8 and calpain-1, unlike a closely related
homologue, PbICP. As discussed earlier unlike PbICP, falstatin
blocks proteolytic activity of caspases and calpain-1 in micro molar
ranges [13]. The possible reasons may be besides it multimeric
forms, falstatin structure is closer to serpins family and seems to
have more flexible loops compare to PbICP. Since falstatin appear
as multimeric forms as compare to PbICP, and thus it may also
interact with caspases and calpains differently. Inhibition of
caspases and calpain-1 by falstatin may be helpful in programmed
cell death and to protect the exposed merozoite from host
proteases like caspases and calpains. Therefore, it will be very
interesting to solve the complex structure of falstatin with cysteine
protease. The structural study of falstatin will open new dimension
in the field of inhibition of proteases by large protein.
Since a wild type peptide based on falstatin BC loop inhibited
the protease activity of recombinant enzymes. We also tested the
activity of the peptides in parasite infected RBC. The post
Hydrogen Bonds are Crucial for the Inhibitory Activity of
Falstatin
The structural analysis of PbICP and chagasin suggested the
importance of the BC loop (LDSVN287GN289GF291VW) in
inhibition of cysteine proteases [12,15]. To understand the
interactions that govern the inhibitory activity of falstatin,
homology modeling energy minimization was carried out. These
studies suggested that Asn289 of falstatin, which lies in the BC loop,
forms hydrogen bonds with Asn21 and Cys22 of VP2, and with
Cys41 of FP3 (Fig. 3) and also hydrophobic shielding between
Phe291 of the inhibitor and the surrounding active site regions of
VP2 and FP3. To analyze the role of predicted hydrogen bonding
and hydrophobic interactions in the BC loop of falstatin, two
mutant
peptides
(LDSVN287GA289GA291VW
and
287
289
291
LDSVN GA GF VW) were synthesized and did hemoglobin hydrolysis assay. Comparison of double (N289A F291A) and
single mutations (N289A) with the wild type peptide, it was
suggested that only asparagine (N289) residue was required for
efficient inhibition (Fig. 4 AB). Since no additive effect of double
mutation was seen, our result suggested that only hydrogen bonds
between the BC loop of falstatin and malarial proteases (VP2, FP3)
are required for efficient inhibition (Fig. 4 AB). Further,
hemoglobin hydrolysis using spectroscopy and SDS-PAGE analysis suggested that the BC loop mutant peptides were slow
inhibitor of VP2 and FP3 compare to wild type peptide (Fig. 4 A–
E).
Effect of the Falstatin BC Loop on P. falciparum
Trophozoites
The entry of fluorescent peptides (mol. wt. range from 653 to
3200 Da) into parasitized red blood cells has been previously
investigated [33] and shown that peptides up to 2365 Da can be
transported into the parasite. Similarly to test the effects of the wild
type BC loop peptide (LDSVN287GN289GF291VW) in intact
parasites, P. falciparum rings were incubated with the wild type and
mutant peptides (LDSVN287GA289GF291VW), and after 30 hours
of incubation, slides were prepared and parasites were examined
(Fig. 6A). There was a clear morphological change in wild type
peptide-treated parasites, with the appearance of a swollen, darkstaining food vacuole, as seen after incubation with the cysteine
protease inhibitor, E-64 (Fig. 6A). To rule out the nonspecific
toxicity effect of the wild type BC loop peptide, equal concentration of a mutant peptide (LDSVN287GA289GF291VW) was added
in the culture as a control (Fig. 6A). This experiment suggested
that the wild type BC loop peptide specifically inhibited the
parasite cysteine proteases. We also estimated parasite production
of free heme, a marker of hemoglobin hydrolysis [31]. We saw a
dose-dependent reduction in free heme formation in peptide
treated compared to untreated parasites (Fig. 6B).
Discussion
We previously showed that falstatin, an endogenous cysteine
protease inhibitor of P. falciparum, appears to play a key role in the
invasion of erythrocytes by merozoites of P. falciparum. Further, the
falstatin homolog PbICP appears to play a key role in hepatocyte
invasion by sporozoites [14]. Our present study focuses on
understanding the interactions responsible for falstatin inhibitory
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Interactions between Cysteine Proteases-Falstatin
incubation of the parasite treated with a peptide showed distinct
food vacuole abnormalities with undegraded hemoglobin, suggested that activities of native cysteine proteases can be blocked by
a BC loop peptide. The incubation of a mutant peptide did not
show similar food vacuole abnormalities suggested that phenotypic
effect was not due to nonspecific toxicity. To further evaluate the
effects of a wild type peptide on an in vitro culture of P. falciparum,
free heme measurement was done. Earlier reports stated that free
heme was generated because of hemoglobin hydrolysis by
proteases, and free heme produced should be directly proportional
to the extent of hemoglobin degradation. A dose-dependent
reduction in free heme formation in peptide treated compared to
untreated parasites further confirmed that a peptide based on BC
loop inhibited the activities of cysteine proteases in P. falciparum
culture.
Designing inhibitors based on protein–protein interactions
(PPIs) is a new approach especially in malaria. A recent study
explained the inhibition of calpain by mimicking a natural proteinprotein interaction between calpain and its endogenous inhibitor,
calpastatin [35]. Furthermore, researchers aimed to block the
interactions of two monomers, and prevent it forming the active
dimer interface, and developed the first small molecule inhibitor of
a herpes virus protease that blocked PPIs [36]. Since falstatin is
released upon schizont rupture, and antibodies that inhibited
falstatin action specifically blocked merozoite invasion of erythrocytes, further suggesting that the inhibitor functions to prevent
inappropriate activity by parasite and/or host cysteine proteases,
and thereby facilitates erythrocyte invasion. Therefore, PPIs
between cysteine proteases and falstatin may be targeted in future.
Designing inhibitors based on PPIs may be less prone to drug
resistant mutation because drug resistance mutation seems less
likely to be tolerated by a complex protein-protein interface.
Although our models predicted that the hydrogen bonds and
hydrophobic interactions between the BC loop and active sites of
malarial cysteine proteases are crucial for strong inhibitory
potency of falstatin. But mutagenesis study further confirmed that
only hydrogen bonds between the BC loop of falstatin and
malarial proteases are required for efficient inhibition. Our results
identify the first biochemical evidence of the crucial interactions
that is governing the functional property of falstatin, and indicates
that falstatin is the first known endogenous inhibitor function as
multimeric forms. The Inhibition of enzymes by BC loop provides
insights into protein-protein interactions during proteases-inhibitor
cross talk. This information can be taken as an interesting starting
point in exploring the mechanism of falstatin function and may be
helpful in drug development against malarial cysteine proteases.
Conclusion
The BC loop of falstatin is a target for mediating inhibition of
cysteine proteases of malaria parasites via hydrogen bonds.
Supporting Information
Figure S1 Expression, purification and refolding of VP2, FP3
and falstatin constructs. A; The wild falstatin and its point
mutations (Asn287 and Phe397) were constructs, expressed and
purified, SDS-PAGE showing different elution of VP2 (B) and FP3
(C), purified by Ni-NTA chromatography using imidazole
gradient.
(TIF)
Effect of falstatin mutants on hemoglobin hydrolysis.
FP3 was incubated with falstatin mutants (Asn287 and Phe397) or E64 and hemoglobin in 100 mM acetate buffer (pH 5.5), and
hemoglobin hydrolysis was assessed with 15% SDS-PAGE.
(TIF)
Figure S2
Acknowledgments
We thank Akansha Pant and Parvathy for technical assistance, Dr.
Byoung-Kuk Na (Gyeongsang National University, South Korea) for his
generous gift of PvPM4, and Dr. Agam Singh (National Institute of
Immunology, New Delhi, India) for his generous gift of native protein
markers. This paper bears the NIMR publication screening committee
approval no. 010/2013.
Author Contributions
Conceived and designed the experiments: S. Sundararaj KCP. Performed
the experiments: S. Sundararaj KCP AKS RS S. Sharma. Analyzed the
data: S. Sundararaj AKS RKD PJR KCP. Contributed reagents/
materials/analysis tools: S. Sundararaj AKS KV AA RKD PJR KCP.
Wrote the paper: KCP PJR S. Sundararaj.
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