International Journal of Applied, Physical and

Bio-Chemistry Research (IJAPBCR)

ISSN(P): 2277-4793; ISSN(E): 2319-4448
Vol. 6, Issue 3, Oct 2016, 1-16
TJPRC Pvt. Ltd.

IN SILICO MODELING AND DOCKING STUDIES OF ABELMOSCHUS

MOSCHATUS TRYPSIN INHIBITOR (AMTI-II)
D. MUNI KUMAR1, K. SANDEEP SOLMON2, K. VIJAYA RACHEL3 & D. SIVA PRASAD4
1
2,3

Post Doctoral Fellow, Department of Biochemistry, Andhra University, Visakhapatnam Andhra Pradesh, India

Assistant Professor, Department of Biochemistry Bioinformatics, GITAM University, Visakhapatnam Andhra Pradesh, India
4

Professor, Department of Biochemistry, Andhra University, Visakhapatnam, Andhra Pradesh, India

ABSTRACT
A potent trypsin inhibitor (AMTI-II) was isolated and purified from the seeds of Abelmoschus moschatus.
Using advanced proteomic techniques like MALDI-TOF, the inhibitor was sequenced and analyzed using MASCOT
software. A 3D model of AMTI-II was constructed using LOMETS, online threading server. The modeled inhibitor
consists of 10 - sheets and a single -helix which was further assessed by PROCHECK and VERIFY-3D programs and
the results support it to be a reliable one. The docked interactions between the predicted structure of AMTI-II and bovine
pancreatic trypsin (3MFJ) were studied using Clus Pro 2.0. Docking studies revealed that the inhibitor binds at
conserved amino acid residues of trypsin with lowest possible energies for both electrostatic and hydrophobic
demonstrated both by in vitro and in silico approaches. Since protease inhibitors are recognized as therapeutic agents for
controlling human diseases, understanding their structure and function is the primary aspect to work for the
development of potential drug with these proteins.
KEYWORDS : Trypsin Inhibitor, Amtsi-II, MALDI-TOF, MASCOT, Threading, Docking

Original Article

interactions thereby inhibiting the catalytic activity of trypsin. The potent inhibitory activity of AMTI-II was

Received: Sep 16, 2016; Accepted: Sep 30, 2016; Published: Oct 05, 2016; Paper Id.: IJAPBCROCT20161

INTRODUCTION
Protease inhibitors, proteins or peptides capable of inhibiting the catalytic activities of proteolytic
enzymes, are widely distributed in nature [1] and in plants, they are abundant in reproductive and storage organs
and vegetative tissues. These inhibitors have been extensively studied because of their applications towards human
welfare. Although earlier workers concentrated on anti-nutritional properties of protease inhibitors, scientists of
late have focused their attention on their potential use as bioinsecticides [2-3], as therapeutic agents for the
treatment of diseases associated with enhanced protease activities including HIV [4-5], cancer [6] and as
antimicrobial agents active against several bacterial and fungal strains [7-9].
Protease inhibitors are classified based on their amino-acid sequences, localization of the reactive sites,
disulfide bridge topology, mechanisms of action, three-dimensional structures and stability [10]. Serine protease
inhibitors (serpins) have been isolated, purified and characterized from a number of plant sources [11-14].
Kunitz type of serpins were reported to be active mainly against trypsin and chymotrypsin [15]. Existence of
multiple molecular forms of protease inhibitors has also been reported in plant tissues [16, 5, 17].
Protease inhibitors serve as tools for the fundamental studies on protein - protein interactions and are used
as models for the study of structure - function relationship. Knowledge of the three-dimensional (3D) structure of
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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

protease-protease inhibitor complex helps in understanding the function of protease inhibitors at the molecular level.
The number of protease inhibitors isolated and identified so far is extremely large and form a good system for studying the
aspects of molecular evolution and structure function relationships. Scientists working in the field of Bioinformatics have
already developed several algorithms for predicting protein-protein interactions [18]. Protein structures can be modeled
either ab initio from sequence alone or by comparative methods that rely on a database of known protein structures
[19-20].
Studies related to the interaction between protease inhibitor and protease, its structure, active site prediction, motif
and domain identification, phylogeny are useful in understanding the potential mechanism of protease inhibitor and
protease binding [21]. An in silico approach need to be followed for predicting the structure of protease inhibitor and study
its interaction with protease.
Hence, in the present study, AMTI-II, the major trypsin inhibitor with associated antimicrobial and lectin
activities from the seeds of Abelmoschus moschatus is further characterized by an In silico approach for its amino acid
composition using MALDI-TOF and MASCOT software and structure prediction by Threading. Phylogenetic analysis was
also made for finding the evolutionary distance between related protease inhibitors. The model is validated and docking
studies are carried out using different algorithms.

MATERIALS AND METHODS

Retrieval of AMTI-II Sequence Using MALDI-TOF
Purified AMTI-II was subjected to MALDI-TOF. The spectrum obtained was analyzed by the online MASCOT
software (http://www.matrix-science.com) to obtain the sequence of the inhibitor [22]. Amino acid sequence of AMTI- II
was

analyzed

for

probable

internal

trypsin

and

chymotrypsin

cleavage

sites

using

Peptide-Cutter

(http://us.expasy.org/tools/peptidecutter/). Polypeptide fragments obtained were compared with the amino acid sequences
of mass ions deduced by MALDI-TOF analysis of purified inhibitor protein.
Sequence Databases
Sequence was retrieved by means of the Sequence Retrieval System (SRS) service at the European Bioinformatics
institute (EBI, http://srs.ebi.ac.uk) [23].
PIR
The Protein Information Resource (PIR) was used to deduce the composition and molecular weight of the
inhibitor.
Phylogenetic Analysis
For constructing phylogenetic tree, Kimura's method [24] was used in the present work. UPGMA (Un-weighted
Pair Group method with Arithmatic Mean) and NJ (Neighbor Joining) and the generated pair-wise alignments were used
for finding the evolutionary distances between related protease inhibitors. Pair-wise distances thus calculated were used to
create a phylogenetic tree employing neighbor-joining (NJ) algorithm [25] with 100 boot strap replicates.

Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

Conserved Domain Database (CDD)

The Conserved Domain Database (CDD) has been used to explicitly define domain boundaries and provide
insights into sequence, structure and function relationships.
Motif Scan
Motif scan has been employed to find out all known motifs that occur in the inhibitor sequence [26].
Protein Modeling by LOMETS (V 3.0)
The

structure

of

AMTI-II

was

predicted

through

LOMETS

(Local

Meta-Threading-Server)

(http://zhang.bioinformatics.ku.edu/ LOMETS), a locally installed meta-server method for protein structure prediction
[27]. The model was further validated by PROCHECK that provides a detailed check on the stereochemistry of a protein
structure [28-29] and Verify3D (http://nihserver.mbi.ucla.edu/Verify 3D/) [30].
Docking Studies
Proteinprotein docking was done by ClusPro 2.0 (http://nrc.bu.edu/cluster) [31] which is a fully automated
web-based program based on CAPRI (Critical Assessment of Prediction of Interactions). A filtering method is applied to
set of structures, selecting those with hydrophobic, good electrostatic and desolvation free energies for further clustering.
The program output is a short list of co-efficient weights of putative complexes ranked according to their clustering scores.
Molegro Virtual Docker (MVD) has been used for versatile visualization of 3D complexes of both protein and ligand
derived from X-ray/NMR or homology modeling [32].

RESULTS
Purification and Characterization of AMTI-II
AMTI-II was purified to apparent homogeneity following ammonium sulphate fractionation, ion-exchange
chromatography on DEAE-cellulose and gel permeation on Sephadex G-100 (33). The molecular weight of AMTI-II as
determined by gel filtration on Sephadex G-200 was 22.4 kDa (Figure 1). This value was similar to the one obtained for the
inhibitor by SDS-PAGE [33].

Figure 1: Molecular Weight Determination of Trypsin

Inhibitors by Gel Filtration on Sephadex G-200
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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

Plot of elution volume against log molecular weight of standard proteins () and AMTI II ()Standard Protein
Markers

Phosphorylase b, 97kDa

Bovine serum albumin, 67kDa

Ovalbumin, 45kDa

Chymotrypsinogen A, 25kDa

Soybean trypsin inhibitor (SBTI), 20.1 kDa

Lysozyme, 14kDa
AMTI-II was then subjected initially to in-gel digestion with trypsin and later with chymotrypsin, and the

resulting peptides were analyzed using MALDITOF. Interpretation of the MALDITOF spectrum was carried out using
the MASCOT software (Figure 2). The MALDITOF analysis indicated identity of AMTI-II to the reported Trypsin
inhibitor A of Glycine max with a Score of 691(Table 1).
The fact that AMTI-II is obtained in pure form is evident from the absence of other prominent peaks in the
spectrum and the calculated mass of it from MALDI-TOF was found to be 18.3 KDa which was closely correlated to that
obtained by SDS-PAGE and gel filtration. From MALDI-TOF analysis, followed by sequential overlapping of both tryptic
and chymotryptic peptides, the amino acid sequence and composition was deduced and the putative sequence of AMTI-II
is as follows:
>Abelmoschus moschatus Trypsin inhibitor (AMTI -II) from Abelmoschus moschatus
VLDNEGNPLGGIRAAPTGNERCPLAAPTGNERCPLTVVQSRNELDKGIGTIISSPYRGIGTIISSPYRFIAEGH
PLSLKRIERVSDDEFNNYIGENKDAMDGWFRDAMDGWFRVSDDEFNNYKLVFCPQQAEDDKCGDIGISIDH
DDGTRRNKPLVVQFQKLDK

Figure 2: MALDI-TOF Spectrum of AMTI- II after in-Gel Digestion. Masses are Expressed in Daltons
Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

The compositional and molecular weight analysis was done by using Protein Information Resource (PIR).
The results showed that there were a total of 164 amino acid residues in

AMTI-II with a molecular weight of 18,304.22

daltons (Figure 3). The inhibitor was rich in aspartic acid, glycine, glutamic acid, asparagine, isoleucine, arginine, leucine
and serine. Moderate amounts of lysine, proline, threonine, valine and tyrosine were observed. The number of acidic amino
acids prevailed over the basic amino acids in AMTI-II.
Table 1: Interpretation of AMTI-II MALDI Data
Fragment
Location
3 11
27-41
55-62
63-71
72-87
77-87
90-100
118-130
131-143
136-143
147-156
157-184
189-202

Resulting Peptide
VLDNEGNPL
GGIRAAPTGNERCPL
AAPTGNER
CPLTVVQSR
NELDKGIGTIISSPYR
GIGTIISSPYR
FIAEGHPLSLK
RIERVSDDEFNNY
IGENKDAMDGWFR
DAMDGWFR
VSDDEFNNYK
LVFCPQQAEDDKCGDIGISIDHDD
GTRR
NKPLVVQFQKLDK

Calculated Mass
(Mascot)

Observed
Mass

969.4767
1567.7889
814.3933
1058.5543
1761.9261
1162.6346
1211.7349
1655.7539
1537.6983
996.4124
1229.5200
3061.7652
1554.7914

969.3007
1568.5294
815.4395
1059.6150
1763.0538
1163.7076
1210.6710
1656.4696
1538.8147
997.4722
1230.5850
3062.2718
1555.49

No match was found to some peptides with various molecular masses like 604.3493, 834.5330, 1013.4634,
1036.6011, 1042.5892, 1200.76102, 1230.5843, 1262.7791, 1351.7061, 1521.8524, 1786.9386, 2455.4272, 2771.5483,
3244.0368 and 3261.0174.

Figure 3: Composition/Molecular Weight Analysis of AMTI-II by PIR

Comparison of AMTI-II with other plant PI sequences by phylogenetic analysis revealed that the inhibitor belongs
to Kunitz type of trypsin inhibitor family (Figure 4).
The conserved domain database, based on protein domain annotation of AMTI-II, identified a similar type of
domain present in Soybean trypsin inhibitor (STI) with E-value of 1.31e-35. This confirms that AMTI-II belongs to Kunitz
family through protein sequence annotation (Figure 5). Motif scan also confirmed that AMTI-II has two different types of
motifs belonging to Kunitz and Thaumatin family.

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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

Motif scan tool has identified three motifs in AMTI-II and the motifs are at amino acid residues located between
10-24, 25-57 and 113-161 and form motifs similar to that of Kunitz family and amino acids present at the positions
120-128 form motifs similar to that of Thaumatin family (Figure 6).

Figure 4: Phylogenetic Tree Analysis of AMTI-II with Serine Protease Inhibitors

Bootstrap values are indicated on the branch points
CYS- Cysteine proteinase inhibitor; PI1- Potato typeI proteinase inhibitor; SPI- Serpin; BRI- Cereal
trypsin/-Amylase inhibitor; KNI- Soy bean trypsin inhibitor (Kunitz);

MSI- Mustard (Sinapsis) trypsin inhibitor.

SQI- Squash inhibitor;; SNTI-Soap nut trypsin inhibitor; BBI- Bowman-Birk serine proteinase inhibitor;
MCI- Metallocarboxypeptidase inhibitor PI2- Potato typeII proteinase inhibitor

Figure 5: Conserved Domains of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

Figure 6: Motif Scan of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

3-D structure of AMTI-II was generated using LOMETS, a threading based secondary structure prediction
method which has initially predicted secondary structure of AMTI-II sequence by PSI-PRED. The modeled AMTI-II
consists of 10 - sheets and a single -helix (Figure 7).
The predicted gene ontology deduced by I-TASSER for the obtained sequence is GO: 0004866 which implies
endopeptidiase inhibitor activity. The molecular function of the protein is GO: 0004867 which infers serine-type
endopeptidase inhibitor activity and GO: 0045735 nutrient reservoir activities.

Figure 7: 3D Structure of AMTI-II after modeling with LOMETS the

-Sheets are Represented by Arrows and -Helix by Cylinder
The structure of AMTI-II was further validated using PROCHECK server to assess possible conformation by
Ramachandran plot. In the plot (Figure 8), the most allowed regions are indicated by red patches, while yellow areas show
allowed regions and has an average allowed number of residues 96.3% within the allowed region and 3.7% of the residues
are in the disallowed region of the plot (Table 2). Assessment and authentication of Ramachandran plot analysis revealed
that the trypsin inhibitor from Abelmoschus moschatus is of good quality, representing the degree of accuracy in the
predicted structure.
The predicted structure was also confirmed by using verify 3D program, which shows the value of 71.52% of
residues with 3D-1D score >0.2 for most of the residues indicating the compatibility of the residues with structure
predicted and the compatibility score above zero corresponded to acceptable side chain environments. The compatibility
score for the whole AMTI-II protein varied from 0.05 to 0.67 (Figure 9).
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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

Figure 8: Ramachandran's Plot of AMTI-II Built Using PROCHECK

Table 2: PROCHECK Results of AMTI-II Residues Falling in Most
Favoured, Additional Allowed, Generously and Disallowed Regions
Amino Acid Residues
Residues in most favoured regions
Residues in additional allowed regions
Residues in generously allowed
Regions
Residues in disallowed regions

80.1
12.5

Number of Amino
Acid Residues
109
17

4.4

2.9

Figure 9: Structure Validation of AMTI-II by VERIFY-3D Graph

Docking Studies
Modeled AMTI-II was docked with bovine pancreatic trypsin (3MFJ) using Cluspro 2.0 and it resulted in flexible
bound docking between the two proteins. Cluspro showed strong electrostatic, hydrophobic and balanced interaction
between AMTI-II and 3MFJ and the results obtained were found to be most promising especially with respect to both
Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

electrostatic and hydrophobic interactions which were visualized using MVD visualizer.
In the electrostatic interactions, specific charge-charge interactions in the binding interface play a vital role.
Electrostatic surface representation and stick model representation for electrostatic interactions of AMTI-II and 3MFJ were
depicted in Figure 10. The residues involved mainly in electrostatic interaction are presented in Table 3. There exists a
good electrostatic interaction between AMTI-II and 3MFJ. The docked complexes elucidate structural details of interaction
and indicate key residues that are involved in the interaction.
Hydrophobic surface representation and Stick model representation for hydrophobic interaction of AMTI-II and
3MFJ were shown in Figure 11. The residues involved mainly in hydrophobic interaction are presented in Table 4.
AMTI-II and 3MFJ also exhibited a strong hydrophobic interaction.

Figure 10: Elecrotstatic Surface Representation for Electrostatic

Interaction of AMTI-II (Left) and 3MFJ (Right)

Figure 11: Hydrophobic Surface Representation for Hydrophobic

Interaction of AMTI-II (left) and 3MFJ (Right)
Table 3: Electrostatic Interactions of AMTI-II and 3MFJ
S. No
1
2
3
4
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AMTI-II
ASN30 (Positive Electrostatic potential)
ARG32 (Positive Electrostatic potential)
ASP101 (Negative Electrostatic potential)
ASN90 (Positive Electrostatic potential)

3MFJ
ALA24 (Negative Electrostatic potential)
GLY23 (Negative Electrostatic potential)
TYR20 (Positive Electrostatic potential)
THR21 (Negative Electrostatic potential)
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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

5
6
7
8

Table 3: Contd.,
SER85 (Negative Electrostatic potential)
LYS188 (Positive Electrostatic potential)
GLU95 (Negative Electrostatic potential)
LYS159 (Positive Electrostatic potential)
LYS135 (Positive Electrostatic potential)
GLU186 (Negative Electrostatic potential)
GLN130 (Negative Electrostatic potential)
LYS222 (Positive Electrostatic potential)

The positive electrostatic potential (NH2+) of LYS159 binds with Negative electrostatic potential (COO-) of
GLU95 to neutralize the negative charge similarly LYS135 binds with GLU186.
Table 4: Hydrophobic Interactions of AMTI - II and 3MFJ
S. No
1
2
3
4
5

AMTI -II
PHE104 ( non-polar)
ALA107 (non-polar)
MET108 (non-polar)
TRP111 (Slightly polar)
PHE126 (non-polar)
PHE89 (non-polar)

3MFJ
TYR59 (polar)
SER61(polar)
GLY62 (non-polar)
LYS109 (polar)
ALA111 (non-polar)

The non-polar side chains of hydrophobic amino acids interact with side chains of polar amino acids to form
hydrophobic interactions.

DISCUSSIONS
Knowledge on the three dimensional (3D) structure of protein is necessary for understanding its function at
molecular level and this current topic of structural biology employs techniques such as X-ray crystallography, nuclear
magnetic resonance (NMR) spectroscopy and electron microscopy for determining the structure of proteins. To be able to
perform their biological function, proteins often fold into one or more specific spatial conformations, driven by a number
of non-covalent interactions such as hydrogen bonding, ionic interactions, Vander Waals' forces and hydrophobic
packing.In silico methods have been useful for predicting 3D structure of binding sites when experimental information is
lacking.
AMTI-II was purified to apparent homogeneity and characterized by wet lab methods and in the present study,
further analysis was carried out using In silico methods. An unambiguous identification of the purified inhibitor was
possible through MALDITOF and analysis of the resultant spectrum, which gave a series of mass ions, corresponding to
tryptic and chymotryptic peptides from the purified inhibitor. On the basis of matching mass ion fragments in the protein
database(s), the MASCOT software assigned high probability of identity to the reported Trypsin inhibitor A of Glycine
max (Score: 691). The MASCOT software was advantageous in that it could account for the carbamidomethyl substitutions
in the chymotryptic fragments. The putative sequence of AMTI-II predicted was composed of single polypeptide chain
with 164 amino acids. The calculated mass from MALDI-TOF was found to be 18.3 KDa excluding the carbohydrate
content and it was closely correlated to that obtained by gel filtration i.e., 22.4 kDa. AMTI-II was found to be a
glycoprotein with neutral sugar content of 4% and was devoid of amino sugars [33] and the deglycosylated inhibitor
retained its maximum trypsin inhibitory activity suggesting that the carbohydrate moieties are not required for its inhibitory
activity [34].
Trypsin inhibitor (MKTI) from the seeds of Murraya koenigii (curry-leaf tree) is a single polypeptide with a
molecular weight of 27 kDa on SDSPAGE [35]. However, on MALDITOF analysis, a molecular weight of 21.4 kDa
was obtained for the protein. No free sulfhydryl groups were detected by Ellmans reagent, but the four cysteine residues
Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

11

identified involve in the formation of two disulfide bridges. AMTI-II after treatment with 2-mercaptoethanol showed a
single sharp band on gels supporting the monomeric nature of the protein.
The amino acid sequence at the putative inhibitory site suggests trypsin and chymotrypsin specificity, since there
are arginine and lysine residues specific towards trypsin; tyrosine, tryptophan and phenylalanine towards chymotrypsin
respectively. Amino acid changes at active site are known to affect specificity of inhibitor towards proteinases and also
might change binding to target enzymes [36].
Proteinase inhibitors of known primary structures are currently grouped into 10 families from sequence
homology. Comparative phylogenetic analysis with 30 other complete coding sequences of protease inhibitors from
dicotyledonous and monocotyledonous plants was done with the inhibitor sequence to probe relationships between
inhibitors and to identify any evolutionary traits. It was observed that the

AMTI-II formed a distinct cluster with the

protease inhibitors from Bauhinia variegata, Erythrina caffra and Glycine max. AMTI-II could be classified into
Kunitz-type trypsin inhibitor family. AMTI-II shared similar structural and functional properties with that of Soybean
trypsin inhibitor (STI), particularly Kunitz type inhibitor. Motif scan results for AMTI-II also revealed that strong matches
were observed with Kunitz type inhibitors legume family.
A 3D model of AMTI-II was constructed using LOMETS. The modeled inhibitor of AMTI-II consists of 10
- sheets and is a single -helix, which is an exception of kunitz type of inhibitors, where absence of -helix is the
characteristic property of those inhibitors.
Kunitz-type serine proteinase inhibitors are the polypeptides with molecular masses varying from 20 - 24 kDa,
with four cysteine residues that form two disulfide bonds, devoid of -helix and a common structural fold composed of a
-trefoil formed by 12 antiparallel -strands with long interconnecting loops presenting one or two reactive sites for serine
proteinases [37-38]. These inhibitors react with their cognate enzyme in a common substrate like standard canonical form
where the inhibitor binds to enzyme through an exposed complex binding loop, complementary to the active site of
enzyme.
LOMETS has been used to generate a structural model of Api m6 from Apis mellifera venom [39] which
exhibited a common fold, which is dominated by an exposed binding loop including the putative protease binding site and
showing a typical canonical conformation that is essential for the biologic activity of protease inhibitors. A model of
Potato virus A VPg has been created by LOMETS and positioned the known functions and structural properties of
potyviral VPgs on the novel structural model which suggested an elongated structure with a hydrophobic core composed of
antiparallel -sheets surrounded by helices and a positively charged contact surface where most of the known activities are
localized [40].
The dependability and quality of this model was ensured by evaluating the backbone conformation, angles and
bond lengths based on Psi/Phi using Ramachandran plot was further assessed by PROCHECK and VERIFY-3D programs
and the results support it to be a reliable one.
Structure modeling and Ramachandran plot analysis were carried out to validate the structural and functional
analysis of cysteine protease and cystatin from tomato [41]. In another study, in silico studies were conducted for structural
modeling of soap nut trypsin inhibitor (SNTI) by Ramachandran plot analysis, and protein models were validated by
computational tools [42]. The 3D structure was predicted by using modeler.
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Pohekar & Chikhale [43] generated

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D. Muni Kumar, K. Sandeep Solmon, K. Vijaya Rachel & D. Siva Prasad

3-D structure of soybean trypsin inhibitor (SBTI) by homology modeling and the resultant structure was evaluated by
PROCHECK, ERRAT and Verify 3D program showing the compatibility of the residues in the structure predicted.
Docking studies with bovine pancreatic trypsin (3MFJ) reveal that the inhibitor binds at the conserved amino acid
residues of trypsin with lowest possible energies for both electrostatic and hydrophobic interactions thereby inhibiting the
catalytic activity of trypsin. There exists a strong electrostatic interaction and hydrophobic interactions between AMTI-II
and 3MFJ.
The protein docking server ClusPro has been participating in critical assessment of prediction of interactions
(CAPRI) since its introduction [44-45]. Protein-protein docking involves matching shape complementarity, some
approaches focus specifically on matching surfaces and others enhance the search for geometric complimentarity by
matching the position of surface spheres and surface nominals [46].
The interaction between AMTI-II and bovine pancreatic trypsin (3MFJ) observed in this in silico analysis with
respect to both electrostatic and hydrophobic is found to be most promising in terms of docking and is useful for
understanding the potential mechanism of inhibiting the enzyme. Further structural studies would help in the validation of
the putative protein.
In our previous study, AMTI-II was shown to exhibit potent antibacterial activity towards Staphylococcus aureus,
Escherichia coli, Proteus vulgaris, Bacillus subtilis, Streptococcus pneumoniae, Bacillus cereus and moderately against
Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae and Streptococcus pyogenes. The trypsin
inhibitor also moderately affected the growth of fungal species, Candida albicans, Candida tropicalis, Asperigillus flavus,
Saccharomyces cerevisiae, Candida glabrata and Asperigellus niger. Results obtained suggest that AMTI-II may serve as
an antimicrobial agent active against pathogenic microbes [47]. AMTI-II is unique in that it exhibited hemagglutinating
activity towards human and animal erythrocytes. Hence, AMTI-II may be useful in the agricultural front for developing
transgenics after carrying out extensive in vitro studies against midgut proteases of insect pests [34].
The potent inhibitory activities of AMTI-II towards Bovine pancreatic trypsin was demonstrated both by in vitro
[48] and in silico approaches. The interaction of AMTI-II with trypsin, its structure, active site prediction, motif and
domain identification, phylogeny proposed in this study are useful for understanding the potential mechanism of the
inhibitor and enzyme binding.

CONCLUSIONS
Since serpins have versatile therapeutic effects in human system with respect to controlling number of diseases,
their interaction with serine proteases will help in understanding their mechanism of inhibition at the molecular level, as
well as protease-protease inhibitor associations. In the present study, a 3D model of AMTI-II was constructed using
LOMETS consisting of 10 - sheets and a single -helix and further assessed by PROCHECK and VERIFY-3D programs
and the results support the model a reliable one. Docking studies reveal that the inhibitor binds at the conserved amino-acid
residues of trypsin with lowest possible energies for both electrostatic and hydrophobic interactions thereby inhibiting the
catalytic activity of trypsin. Hence, the potent trypsin inhibitory activity of AMTI-II was demonstrated by in vitro and in
silico approaches. Identifying the reactive site loop in AMTI-II having trypsin inhibitory activity will be further assessed
and studies related to the structural homology of AMTI-II with other trypsin inhibitors could be helpful in the development
of novel drugs for the treatment of human diseases.
Impact Factor (JCC): 2.6392

Index Copernicus Value (ICV): 6.1

In Silico Modeling and Docking Studies of Abelmoschus Moschatus Trypsin Inhibitor (AMTI-II)

13

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Haq, S.K., Atif, S.M. and Khan, R.H. (2004). Protein proteinase inhibitor genes in combat against insects, pests and
pathogens: natural and engineered phytoprotection. Arch Biochem Biophys, 431,145-159.

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Abdeen, A., Virgos, A., Olivella, E., Villanueva, J., Aviles, X. and Gabarra R, Prat S. (2005). Multiple insect resistance in
transgenic tomato plants over-expressing two families of plant proteinase inhibitors. Plant Mol. Biol, 57(2), 189-202.

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Asztalos Bela, F., Ernst Schaefer, J., Katalin Horvath, V., Caitlin Cox, E., Sally Skinner, Jul Gerrior., Sherwood Gorbach, L.
and Christine Wanke. (2006). Protease inhibitor-based HAART, HDL, and CHD-risk in HIV-infected patients.
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