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HOMOLOGY MODELING AND DOCKING STUDIES ON LUKOCIDIN LUKD
IN STAPHYLOCOCCUS AUREUS
ABSTRACTStaphylococcus aureus(Gram-positive) is a one of the bacterial species and it is frequently
found as part of the normal skin flora on the skin and nasal passages that can cause a wide variety of
infections in humans and other animals through either toxin production or invasion. Over 30
different types of Staphylococci can infect humans, but most infections are caused by
Staphylococcus aureus. It can host phages, such as the Panton-Valentine leukocidin, that increase its
virulence. S. aureus can infect other tissues when barriers have been breached. Its incidence ranges
from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. Existing drugs are
not efficient to control the staph infections completely. So, existing antibacterial compounds are
downloaded from PubChem compound database and docked with the target protein retrieved from
UniProt database. Before docking template protein covalent S-F heterodimer of staphylococcal
gamma-hemolysin is identified by BLASTP. Our target protein is included for homology modelling
using Swissmodel, EsyPred3D,Phyre2 and modeller. Swiss model,EsyPred3D,Phyre2 tools that are
freely accessible from web workspace. By considering the PROCHECK and RMSD values Swiss
model analysis gave the best results than other methods. Q site finder was used to identify the
possible ligand binding sites with coloured regions,amino acid residues and residue numbers.
Protein structure file and ligand files are prepared by the use of ArgusLab. Then alternatively
Protein is docked with different antibacterial compounds. Finally number of interactions and
interaction distance also calculated for those docked compounds by PyMol viewer. Based on this
study, Diathymosulfone, Myrophine, Andrimid, Beclobrate, Probucol compounds are identified as
best antibacterial compounds tht can have the power to kill, inhibit the effect and slows down the
growth of the disease causing Staphylococcus aureus.
1
INTRODUCTION2. Introduction:
Staphylococcus aureus is a bacterial species named from Greek meaning the "golden
grape-cluster berry” Also known as "golden staph" and Oro staphira, it is a facultative anaerobic
Gram-positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin
and nasal passages. S. aureus was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir
Alexander Ogston in pus(An abscess is an enclosed collection of liquefied tissue, known as pus,
somewhere in the body. It is the result of the body's defensive reaction to foreign material. ) from
surgical abscesses. Each year some 500,000 patients in American hospitals contract a
staphylococcal infection Staphylococcus aureus is the most common etiologic agent of skin
abscesses. The regional rate of methicillin-resistant S. aureus (MRSA) abscesses may reflect the
prevalence of local community-acquired MRSA (CAMRSA). EpiInfo software was used to analyze
the antimicrobial susceptibilities of all isolates and the trends in the rates of MRSA. positive
bacteria which includes several species that can cause a wide variety of infections in humans and
other animals through either toxin production or invasion. Staphylococcal toxins are a common
cause of food poisoning, as they can be produced in improperly-stored food.
S. aureus Strains are responsible for food poisoning through the production of an
enterotoxin and pathogenicity is also associated with coagulase positivity. S.aureus may occur as a
commensal on human skin; it also occurs in the nose frequently (in about a third of the population)
and throat less commonly. Over 30 different types of Staphylococci can infect humans, but most
infections are caused by Staphylococcus aureus. The occurrence of ''S. aureus'' under these
circumstances does not always indicate infection and therefore does not always require treatment
(indeed, treatment may be ineffective and re-colonization may occur). It can survive on
domesticated animals such as dogs, cats and horses, and can cause bumblefoot in chickens. It can
survive for some hours on dry environmental surfaces, but the importance of the environment in
spread of ''S. aureus'' is currently debated. It can host phages, such as the Panton-Valentine
leukocidin, that increase its virulence. S. aureus can infect other tissues when barriers have been
2
breached (e.g., skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection
of furuncles). In infants ''S. aureus'' infection can cause a severe disease Staphylococcal scalded
skin syndrome (SSSS).
Staphylococci can be found normally in the nose and on the skin (and less commonly in
other locations) of around 25%-30% of healthy adults and in 25% of hospital workers. In the
majority of cases, the bacteria do not cause disease. However, damage to the skin or other injury
may allow the bacteria to overcome the natural protective mechanisms of the body, leading to
infection. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples,
impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses,
to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock
syndrome (TSS), bacteremia, and sepsis. Its incidence ranges from skin, soft tissue, respiratory,
bone, joint, endovascular to wound infections. It is still one of the five most common causes of
nosocomial infections and is often the cause of postsurgical wound infections. Each year, some
500,000 patients in American hospitals contract a staphylococcal infection.
The occurrence of S. aureus under these circumstances does not always indicate infection
and, therefore, does not always require treatment (indeed, treatment may be ineffective and
recolonisation may occur). It can survive for hours to weeks, or even months, on dry environmental
surfaces, depending on strain. It can host phages, such as Panton-Valentine leukocidin, that increase
its virulence.When the bacteria enter the bloodstream and spread to other organs, a number of
serious infections can occur. Spread of the organisms to the bloodstream is known as bacteremia or
sepsis. Staphylococcal pneumonia predominantly affects people with underlying lung disease and
can lead to abscess formation within the lungs. Infection of the heart valves (endocarditis) can lead
to heart failure. Spread of Staphylococci to the bones can result in severe inflammation of the bones
known as osteomyelitis. When Staph bacteria are present in the blood, a condition known as
staphylococcal sepsis (widespread infection of the bloodstream) or staphylococcal bacteremia
exists. In breastfeeding women, Staph can result in mastitis (inflammation of the breast) or in
abscess of the breast. Staphylococcal breast abscesses can release bacteria into the mother's milk.
Toxic shock syndrome is an illness caused by toxins secreted by Staph aureus bacteria
growing under conditions in which there is little or no oxygen. Toxic shock syndrome is
characterized by the sudden onset of high fever, vomiting, diarrhea, and muscle aches, followed by
low blood pressure (hypotension), which can lead to shock and death.
3
Leukocidin LukD is a protein compound occurred in Staphylococcus aureus Bacteria.
Sequence length of the protein is 327 AA . LukD defines gene name of the Leukocidin protein.
Staphylococcus aureus is an important human pathogen that expresses a variety of exoproteins.
These include the synergohymenotropic toxins (SHTs), which damage host cell membranes by the
synergistic action of two classes of nonassociated proteins, designated S and F. The SHT family is
comprised of the Panton-Valentine leukocidin (PVL), gamma-hemolysin, and other leukocidins
(e.g., LukE, LukD, and LukM). The F components (SHT-F: LukF-PV, LukD, and Hlg-B) and the S
components (SHT-S: LukS-PV, LukE, Hlg-A, Hlg-C, and LukM) of SHT share 70 to 80% and 60
to 80% sequence identity, respectively. Each combination of S and F components is considered to
be a biologically distinct toxin and was shown to differ in their toxicity against leukocytes in vitro
and rabbits in vivo. Neither S nor F component alone is cytotoxic, but components together are
active. These include Panton-Valentine leukocidin (PVL), gamma-hemolysin, LukE-LukD and
others.
Panton-Valentine leukocidin (PVL) is a cytotoxin—one of the β-pore-forming toxins. The
presence of PVL is associated with increased virulence of certain strains (isolates) of
Staphylococcus aureus. It is present in the majority of community-associated Methicillin-resistant
Staphylococcus aureus (CA-MRSA) isolates studied and is the cause of necrotic lesions involving
the skin or mucosa, including necrotic hemorrhagic pneumonia. PVL creates pores in the
membranes of infected cells. PVL is produced from the genetic material of a bacteriophage that
infects Staphylococcus aureus, making it more virulent Panton-Valentine leukocidin (PVL)-
producing Staphylococcus aureus is associated with a broad spectrum of diseases, ranging from
common uncomplicated soft tissue infections to severe diseases such as complicated soft tissue
infections, extensive bone and joint infections, and necrotising pneumonia. Specialised management
of infection based on the presence of PVL may not be required for mild infections, whereas it could
be lifesaving in other settings. Moreover, most severe PVL diseases are recently identified entities
and a 'gold standard' treatment from comparatives studies of different therapeutic options is lacking.
Thus, recommendations are based on expert opinions, which are elaborated based on theory, in vitro
data and analogies with other toxin-mediated diseases.
4
Methicillin-resistant Staphylococcus aureus, known as MRSA, is a type of Staphylococcus
aureus that is resistant to the antibiotic methicillin and other drugs in the same class, including
penicillin, amoxicillin, and oxacillin. MRSA is one example of a so-called "superbug," an informal
term used to describe a strain of bacteria that has become resistant to the antibiotics usually used to
treat it. MRSA first appeared in patients in hospitals and other health facilities, especially among
the elderly, the very sick, and those with an open wound (such as a bedsore) or catheter in the body.
In these settings, MRSA is referred to as health care-associated MRSA (HA-MRSA). The
transmission of MRSA is largely from people with active MRSA skin infections. MRSA is almost
always spread by direct physical contact and not through the air. Spread may also occur through
indirect contact by touching objects (such as towels, sheets, wound dressings, clothes, workout
areas, sports equipment) contaminated by the infected skin of a person with MRSA. More recently,
strains of Staph aureus have been identified that are resistant to the antibiotic vancomycin
(Vancocin), which is normally effective in treating Staph infections. These bacteria are referred to
as vancomycin-intermediate-resistance S. aureus (VISA) and vancomycin-resistant Staph aureus
(VRSA).
Since the Staphyloccocus aureus infection forms resistance to existing antibitics finding
newer antibiotic compounds against staphylococcus aureus can be useful to prevent from Staph
infections. It is more effective to find the drugs or antibiotic compounds against the disease causing
organism by homology modeling and docking. Through homology modeling and docking, we can
find the best compounds working against the disease causing organism. The identified compound(s)
will leads to potent drug(s) against the diseases caused by Staphylococcus aureus.
Thus by using different tools, softwares, available information and databases, the present
study related to staphylococcus aureus(staph infections) have been designed on the basis of
homology modeling and docking with the following objectives.
5
REVIEW OF LITERATURE 3. Review of Literature:
Leukocidin LukD is a protein compound occurred in Staphylococcus aureus Bacteria.
Sequence length of the protein is 327 AA . LukD defines gene name of the Leukocidin protein.
Staphylococcus aureus is an important human pathogen that expresses a variety of exoproteins.
These include the synergohymenotropic toxins (SHTs), which damage host cell membranes by
the synergistic action of two classes of nonassociated proteins, designated S and F (Prevost et al.,
1995) .
The F components (SHT-F: LukF-PV, LukD, and Hlg-B) and the S components
(SHT-S: LukS-PV, LukE, Hlg-A, Hlg-C, and LukM) of SHT share 70 to 80% and 60 to 80%
sequence identity, respectively. Each combination of S and F components is considered to be a
biologically distinct toxin and was shown to differ in their toxicity against leukocytes in vitro
and rabbits in vivo (Gravet et al., 1998). Neither S nor F component alone is cytotoxic, but
components together are active. These include Panton-Valentine leukocidin (PVL), gamma-
hemolysin, LukE-LukD and others (Morinaga et al., 2003).
S. aureus can cause a range of illnesses, from minor skin infections, such as pimples,
impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and
abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis.It is still one of the five most
common causes of nosocomial infections and is often the cause of postsurgical wound infections.
Each year, some 500,000 patients in American hospitals contract a staphylococcal infection
(Bowersox and John, 1999) . S. aureus may occur as a commensal on skin; it also occurs in the
nose frequently (in about a third of the population) and the throat less commonly (Whitt et al.,
2002) .
6
The occurrence of S. aureus under these circumstances does not always indicate infection
and, therefore, does not always require treatment (indeed, treatment may be ineffective and
recolonisation may occur). It can survive on domesticated animals, such as dogs, cats, and
horses, and can cause bumblefoot in chickens. It can survive for hours to weeks, or even months,
on dry environmental surfaces, depending on strain (Cimolai, 2008).
Panton-Valentine leukocidin is a cytotoxin-one of the β-pore-forming toxins. The
presence of PVL is associated with increased virulence of certain strains (isolates) of
Staphylococcus aureus. It is present in the majority of community-associated Methicillin-
resistant Staphylococcus aureus (CA-MRSA) isolates studied and is the cause of necrotic lesions
involving the skin or mucosa, including necrotic hemorrhagic pneumonia (Curran and Al-Salihi,
1980; Szmiegielski et al., 1999; Kaneko and Kamio 2004). PVL creates pores in the membranes
of infected cells. PVL is produced from the genetic material of a bacteriophage that infects
Staphylococcus aureus, making it more virulent (Lina G et al., 1999). Most severe PVL diseases
are recently identified entities and a 'gold standard' treatment from comparatives studies of
different therapeutic options is lacking. Thus, recommendations are based on expert opinions,
which are elaborated based on theory, in vitro data and analogies with other toxin-mediated
diseases (Gillet et al., 2011).
7
AIMS &
OBJECTIVES
4. Aims and objectives:
o To predict a suitable template for Leukocidin (LukD) by performing BLAST search
.
o To perform Homology Modeling for Leukocidin D through various servers and to
compare their results
o To predict the Binding Sites for the protein using Q site finder and to perform
docking with Arguslab.
8
Materials & Methods
5. Materials and Methods:
Data Source:
5.1 UniProt:
The mission of UniProt is to provide the scientific community with a comprehensive, high-
quality and freely accessible resource of protein sequence and functional information(
http://www.uniprot.org/).
The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence
and annotation data.
TOOLS USED FOR HOMOLOGY MODELING AND DOCKING:
5.2. BLAST (Basic Local Alignment Search Tool):
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between
sequences. The program compares nucleotide or protein sequences to sequence databases and
calculates the statistical significance of matches. BLAST can be used to infer functional and
evolutionary relationships between sequences as well as help identify members of gene families
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Basic Local Alignment Search Tool (BLAST) is a
sequence similarity search program. The public interface of BLAST,
http://www.ncbi.nlm.nih.gov/blast, at the NCBI website has recently been reengineered to improve
usability and performance. Target protein fasta sequence is uploaded in the Protein BLAST query
box. Switch the BLAST option to run the program. It Analyses with other protein sequences gives
9
the result by maximum identity. Maximum identity protein is selected for the template
protein(2QK7_B) with 77% identity.
5.3. PDB (Protein Data Bank):
The Protein Data Bank (PDB) archive is the single worldwide repository of information
about the 3D structures of large biological molecules, including proteins and nucleic acids. These
are the molecules of life that are found in all organisms including bacteria, yeast, plants, flies, other
animals, and humans. Understanding the shape of a molecule helps to understand how it works.
This knowledge can be used to help deduce a structure's role in human health and disease, and in
drug development. The most recent release is time stamped and linked on every page in the top
right header. In 2003, the wwPDB was formed to maintain a single PDB archive of macromolecular
structural data that is freely and publicly available to the global community. It consists of
organizations that act as deposition, data processing and distribution centers for PDB data. The
Protein Data Bank is the single worldwide archive of primary structural data of biological
macromolecules. Many secondary sources of information are derived from PDB data. It is the
starting point for studies in structural bioinformatics. Template Protein is Retrieved from PDB.
Fasta sequence and structure of the template protein is download from PDB.
5.4. Swiss Model:
SWISS-MODEL is a fully automated protein structure homology-modeling server,
accessible via the ExPASy web server, or from the program DeepView (Swiss Pdb-Viewer). The
purpose of this server is to make Protein Modelling accessible to all biochemists and molecular
biologists worldwide. Homology models of proteins are of great interest for planning and analysing
biological experiments when no experimental three-dimensional structures are available. Building
homology models requires specialized programs and up-to-date sequence and structural databases.
Integrating all required tools, programs and databases into a single web-based workspace facilitates
access to homology modelling from a computer with web connection without the need of
downloading and installing large program packages and databases . Enter the protein into query and
result will come to your mail. 10
5.5. EsyPred3D:
Homology or comparative modeling is currently the most accurate method to predict the
three-dimensional structure of proteins. It generally consists in four steps: (1) databanks searching
to identify the structural homolog, (2) target-template alignment, (3) model building and
optimization, and (4) model evaluation. The target-template alignment step is generally accepted as
the most critical step in homology modeling . If we submit the query protein they will send result to
our mail.
5.6. Phyre2:
Phyre2 (Protein Homology/AnalogY Recognition Engine) are web-based services for
protein structure prediction that are free for non-commercial use. Phyre2
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) is among the most popular methods
for protein structure prediction having been cited over 1000 times. Like other remote homology
recognition techniques , it is able to regularly generate reliable protein models when other widely
used methods such as PSI-BLAST cannot. Phyre2 has been designed (funded by the BBSRC) to
ensure a user-friendly interface for users inexpert in protein structure prediction methods.
5.7. Modeller:
MODELLER is used for homology or comparative modeling of protein three-dimensional
structures. The user provides an alignment of a sequence to be modeled with known related
structures and MODELLER (http://salilab.org/modeller/) automatically calculates a model
containing all non-hydrogen atoms. MODELLER is available for download for most Unix/Linux
systems, Windows, and Mac.
5.8. PubChem:
The PubChem BioAssay summary pages now display the Gene Ontology (GO)
11
classification of the gene/protein target(s) that were tested by the
bioassay(http://pubchem.ncbi.nlm.nih.gov/). All GO terms that apply to the gene(s)/protein(s) tested
by the bioassay are shown in the GO hierarchy, including: (1) biological processes, (2) cellular
components, and (3) molecular functions. Clicking on any GO term in the hierarchy will retrieve all
bioassays that have tested a protein(s) associated with that term. In addition, the PubChem
BioAssay data summary for a compound now displays a graphical summary of the bioassays that
have tested the compound, categorizing the bioassays by: (1) bioactivity outcomes (active, inactive,
inconclusive, unspecified); (2) top targets; (3) bioactivity types (IC50, EC50, Potency, Ki, etc.); (4)
bioassay types (screening, confirmatory, summary, other). Antibacterial bacterial compounds
downloaded from PubChem. Compounds are downloaded in SDF form. 174 compounds are
downloaded in SDF form from PubChem Database. After then compounds are changed in MOL
files. Downloaded compounds are used in Docking with the protein.
5.9. Q – Site Finder:
Q-Site Finder is a new method of ligand binding site prediction. It works by binding
hydrophobic (CH3) probes to the protein, and finding clusters of probes with the most favorable
binding energy. These clusters are placed in rank order of the likelihood of being a binding site
according to the sum total binding energies for each cluster. Identifying the location of ligand
binding sites on a protein is of fundamental importance for a range of applications including
molecular docking, de novo drug design and structural identification and comparison of functional
sites. It uses the interaction energy between the protein and a simple van der Waals probe to locate
energetically favorable binding sites. Energetically favorable probe sites are clustered according to
their spatial proximity and clusters are then ranked according to the sum of interaction energies for
sites within each cluster (ref 4). CHIME Interface explains some other features with the protein. In
Q-Site Finder Predicted binding site selection is colour-coded according to the likelihood of being
an actual binding site. Green is the most likely, followed by blue, purple and orange/brown. The
atom number, atom type, residue name, chain identifier and residue number are given.
SOFTWARES USED:
5.10. ArgusLab:
12
ArgusLab ( http://www.arguslab.com/arguslab.com/ArgusLab.html ) is a molecular
modeling, graphics, and drug design program for Windows operating systems. It’s getting a little
dated by now, but remains surprisingly popular. To date, there are > 20,000 downloads. ArgusLab
is freely licensed. Arguslab requires a protein and it has various options (Clean geometry, add
hydrogen, delete hydrogen, etc.). Arguslab downloaded from online and installed in our system. It
is available for windows,ubuntu and other operating system users.
5.11. PyMOL:
PyMOL is a user-sponsored molecular visualization system on an open-source
foundation(http://www.pymol.org/). PyMOL today will yield lasting benefit thank to PyMOL's
cross−platform support and open−source license, which guarantee that versions of PyMOL will
remain available to you in the future, regardless of your computer's operating system, your financial
resources, or where your career takes you. Also available by users Choice (for students and
teachers). Students can get by register. They will send a username and password to your mail to
access the PyMol.
METHODOLOGY:
5.12. Retrieval of the target protein sequence:
The protein sequence of Leukocidin D of Staphylococcus aureus was retrived from the
UNIPROT (Entry: A6QI08) (http://www.uniprot.org/) sequence database. It was found out that the
three dimensional structure of Leukocidin D of Staphylococcus aureus is not available in PDB
(http://www.rcsb.org/) database, hence an attempt has been made in the present study to predict the
structure using various servers. Leukocidin D was 327 AA in length.
5.13. Template retrieval:
The NCBI BLAST was used to identify the template for modeling the three dimensional
structure of Leukocidin D of Staphylococcus aureus. The results yielded by NCBI BLAST against
the PDB database revealed that Chain B, A covalent S-F heterodimer of staphylococcal gamma-
hemolysin (PDB ID: 2QK7_B) as a suitable template based on the percentage identity and sequence
13
similarity. Once sequence and template for the target protein was obtained, alignment was
performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The BLAST result
reveals that the target and the template shows Query coverage of 90%, Maximum identity of 77% .
5.14. Homology Modeling:
The three dimensional structure of the protein Leukocidin D is modeled using four different
comparative modeling programs: MODELLER9.10 a freely available computer program, used for
modeling proteins tertiary structure, SWISS MODEL (http://swissmodel.expasy.org/) is a fully
automated protein structure homology-modeling serve, EsyPred3D
(http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/) is a powerful automated
homology modeling program because it performs alignment using neural networks and Phyre2
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) since there is no crystal structure
available in PDB.
5.15. Structure refinements:
The rough model generated was subjected to energy minimization using the steepest descent
technique to eliminate bad contacts between protein atoms. The energy minimization will be
implemented through Swiss-Pdb Viewer (http://expasy.org/spdbv/).
5.16. Validation:
The three dimensional structure of Leukocidin D predicted by using the four different
servers is then evaluated for its stability by using two different servers: ProSA (Protein structure
analysis) (https://prosa.services.came.sbg.ac.at/prosa.php) is an interactive web server used for the
identification of energy profile of the newly predicted protein and SAVS (Structural analysis and
verification server) (http://nihserver.mbi.ucla.edu/SAVES/) have five different maneuvers 14
PROCHECK, WHAT_CHECK, ERRAT, VERIFY_3D, PROVE to validate the given protein
structure .It also afford Ramachandran plot value for the given structure.
5.17. Active Site Prediction:
After predicting the final new model, the possible binding sites for the present in Leukocidin
D were obtained using Q-Site Finder (http://www.modelling.leeds.ac.uk/qsitefinder//). Q-Site
Finder have predicted 16 binding sites in Leukocidin D. These binding sites were compared to the
active site of the template to determine the residues forming the binding pocket. The results
obtained were further used to analyse the protein-ligand interaction.
5.18. Molecular docking:
The binding ability of various ligand compounds in the active site of the protein Leukocidin
D were predicted using ARGUS LAB. The active site residues were selected and the area to be
docked was enclosed by generating a grid box. 173 antibacterial ligand compounds were collected
from the literatures. The 3D structures of all this compounds were obtained from pubchem database.
Docking was performed by interacting all these compounds to the active site of Leukocidin D. The
final results of docking gives information whether this ligand compounds can bind to protein. The
results were inferred based on the docking energy and hydrogen bond interaction.
15
Results & Discussion
6. Results and Discussion:
6.1. Sequence analysis:
The three dimensional structure for a protein can be generated only when the target
and template shares high percentage of similarity. The similarity of the target to its template
sequence should be 30%. Through blast search, the A covalent S-F heterodimer of staphylococcal
gamma-hemolysin Chain B was obtained as a suitable template with the Query coverage of 90%,
Maximum identity of 77% This proves that 2qk7 is a suitable template for Leukocidin D.
6.2. Multiple sequence alignment:
The homology modeling for a protein can be achieved only based on the sequence similarity
between the target and the template protein. The multiple sequence alignment was performed using
ClustalW (Figure 1) which performs the similarity between given sequences.
6.3. Homology modelling:
The absence of the three dimensional structure of Leukocidin D in PDB provoked us to
construct the 3Dstructure for the protein. The three dimensional structure provides valuable insight
into molecular function and also enables the analyses of its interactions with suitable inhibitors.
Eight structures were generated through four different servers EsyPred3D, Swiss model, Modeller
and Phyre2. Finally the structure generated through swiss model (Figure 2) was as suitable model
based upon the Ramachandran plot and visualisation of the superimposition (Figure 3) between
16
target and the template. The RMSD deviation for this structure is 0.077Å which is also low
compared to other predicted models.
Figure 1: ClustalW Result showing sequence alignment
17
Figure 2: Three dimensional structure of Leukocidin D predicted using Swiss model server. Alpha
helixes are given in blue coloue, beta sheets in violet and loops are represented in maroon colour.
6.4. Validation of the structure:
The stereochemistry of the constructed structure of Leukocidin D was subjected to energy
minimization and the stereo chemical quality of the predicted structure was assessed. The
Ramachandran plot for the model showed 82.7% of the residues in the core region, 11.7% residues
in the additionally allowed regions, 0.8% of residues in the generously allowed region and 0.4%. in
the disallowed region (Figure 4 ) were given in Table 2. The overall verify 3D quality was 98.66%
and the ERRAT value was 83.394. Further the energy of the protein obtained by ProSA (Figure 5a
and 5b) was -5.06 which shows Z score of -5.06 .The results obtained were given in Table 1.
Table 1: Validation performed for the structures predicted using various servers for Leukocidin D
S. TOOLS SAVS ProSA Rmsd
18
NO Z score
CORE ADD
ALLOWED
GEN
ALLOWED
DIS
ALLOWED
ERRAT VERIFY 3D
VERIFY 3D
pass/fail
1 Swiss model 87.2% 11.7% 0.8% 0.4% 83.394 98.66% Pass -5.06 0.077 2 EsyPred3D 89.3% 8.4% 1.9% 0.4% 72.242 93.27% Pass -5.54 0.165
3 Phyre2 88.3% 10.9% 0.4% 0.4% 91.065 93.67% Pass -5.08 0.593
4
MODELERmodel 1
89.3% 9.3% 0.7% 0.7% 61.465 83.54% Pass -4.89 0.490
Model 2 87.6% 10.3% 1.0% 1.0% 55.172 83.32% Pass -4.47 0.479
Model 3 90.7% 7.6% 1.0% 0.7% 61.935 82.62% Pass -3.92 0.503
Model 4 89.7% 8.6% 0.7% 1.0% 60.188 88.11% Pass -4.24 0.436
Model 5 89.7% 7.2% 1.4% 1.7% 57.547 86.59% Pass -3.37 0.353
19
Figure 3: Superimposition between Leukocidin D and its A covalent S-F heterodimer of
staphylococcal gamma-hemolysin. The target structure is given in green colour and the
template in red colour.
20
Figure 4: The Ramachandran plot of Leukocidin D predicted using PROCHECK server
-Figure 5a: The ProSA result of Leukocidin D which shows the Z score of the protein.
21
Figure 5b: The dark green line represents the energy of Leukocidin D and the light green line represents the energy of a covalent S-F heterodimer of staphylococcal gamma-hemolysin
6.5. Binding sites prediction using by Q site finder:
Q site finder is online tool used for find the possible ligand binding sites in the target
protein. Qsite finder works by binding hydrophobic (CH3) probes to the protein, and finding
clusters of probes with the most favorable binding energy. Target protein is uploaded in Q site query
box to find the ligand binding sites prediction. It shows possible ligand binding sites and Q site
finder CHIME informations explains some other features with the protein. In Q-Site Finder
Predicted binding site selection is colour-coded according to the likelihood of being an actual
binding site. Green is the most likely, followed by blue, purple and orange/brown. The atom
number, atom type, residue name, chain identifier and residue number are given. Through the
results from Q site finder Possible ligand binding sites are predicted with the residue name and
numbers (ARG 226,ASN 228,GLN 154,GLU 155,HIS 219,ILE 179,LEU 223,LEU 224,LYS
153,LYS 178 MET 180,PHE 116,PRO 222,SER 156,TYR 157). The following figure shows the
result page from the Q site finder.
22
Figure 6: Active site regions present in Leukocidin D
6.6. Docking used by ArgusLab and PyMOL:
ArgusLab is a molecular modeling, graphics, and drug design program for Windows
operating systems and freely licensed. Target protein was uploaded in ArgusLab.The ligand
compounds where then taken one by one for interacting with Leukocidin D. After ligand file
preparation docking energy was calculated by dock a ligand. Docking energy were noted in the
dockin Table 2. The interaction between the ligand and the protein were then viewed in PyMol
viewer. Diathymosulfone and Myophine are the two top most predicted ligands with high docking
energy. Andrimid, Beclobrate, Probucol are the other three best compounds next to the top two
compounds.
Table 2: Docking energy and hydrogen bond interaction of the retrived ligand compounds
with Leukocidin D23
S.No Ligand name Docking energy
No of interactions
Interacting residues+distance
1. Diathymosulfone -12.4494 2 LYS 178(2.4),GLU 155(2.8)
2. Myrophine -11.3731 1 SER 156(2.4)
3. Andrimid -10.6812 3 MET 180(3.0),GLU 155(2.4)(2.0)
4. Beclobrate -10.4707 2 ASN 230(2.6)(2.7)
5. Probucol -10.1249 0
6. Bucloxic Acid -10.0781 3 HIS 219(2.6),ARG 226(2.6),SER 156(2.8)
7. Clomestrone -9.97522 0
8. Feprazone -9.86751 1 SER 156(2.3)
9. Triparanol -9.76569 4 ARG 226(2.3)(3.0)(3.1),LEU 223(3.1)
10. Fenbufen -9.7074 5 LYS 153(2.8),ASN 230(2.4) (2.9)(2.9) (3.0)
11. Thiamphenicol -9.6736 6 ASN 230(2.4)(2.5)(2.7),GLU 155(2.2),LEU 224(2.4),LEU 223(2.3)
12. Doxycycline -9.52488 3 ASN 230(2.5),ASN 228(2.8),SER 227(3.0)
13. Oxamethacin -9.47128 3 ARG 226(3.2)(2.7)(2.2)
14. Rolitetracycline -9.42403 5 HIS 219(3.4),MET 180(2.6),SER 156(2.4)(2.7)(3.0)
15. Ticarcillin -9.39646 7 ARG 226(3.0)(3.0),MET 221(2.1),LEU 223(2.5)(2.6)(2.7),LEU 224(2.9)
16. Torsernide -9.38191 5 SER 156(2.3),HIS 219(3.5),ARG 226(3.0),MET 221(3.2),LEU 223(2.9)
17. Fendosal -9.35108 1 GLN 220(1.9)
18. Sulfacytine -9.34584 4 MET 221(2.4),ARG 226(2.7),ASN 228(1.9),GLU 155(2.0)
19. Clidanac -9.28833 2 MET 180(2.5),SER 156(2.1)
20. Methacycline -9.26672 4 ASN 182(1.7),MET 180(2.7),LYS 178(1.9),SER 156(2.1)
21. Chloramphenicol -9.26395 5 ASN 230(2.9)(2.9),LYS(3.0),GLU 155(2.2),LEU 223(2.7)
24
22. Haloxazolam -9.20491 2 GLU 155(2.6)(3.0)
23. Dicloxacillin -9.18185 3 HIS 219(1.9),ARG 226(3.0),GLU 155(2.3)
24. Carbenicillin -9.17821 4 GLN 220(3.5),MET 221(2.1),GLU 155(2.1),LEU 223(2.4)
25. Tetracycline -9.16807 2 ARG 226(2.8),SER 227(2.9)
26. Flunitrazepam -9.14276 4 ARG 226(2.6)(3.1),LYS 153(3.0),GLU 155(3.3)
27. Talastine -9.10116 0
28. Penicillin G -9.0842 4 GLU 155(2.0),LEU 223(2.2),MET 221(2.0),GLN 220(3.4)
29. Sulfachlorpyridazine -9.07008 5 GLU 155(2.8),LYS 153(3.3),ASN 230(2.7)(3.0),ASN 228(3.4)
30. Nicoclonate -9.07003 1 ASN 230(2.4)
31. Ampicilin -9.06581 3 LEU 223(2.3),GLU 155(2.3)(2.1)
32. Mecloxamine -9.0571 1 SER 156(2.8)
33. Simvastatin -9.05074 3 SER 156(2.8)(2.5),MET 180(3.0)
34. Diclofenac -8.99543 2 GLU 155(2.2),ARG 226(3.0)
35. Temazepam -8.9885 2 MET 180(3.0),LYS 178(2.3)
36. Sulfaethidole -8.9672 3 GLU 155(2.6)(2.4)(2.7)
37. Sulfaproxyline -8.96598 5 PHE 116(3.5),GLU 155(1.9)(2.4),LEU 223(2.9),ASN 228(2.2)
38. Metiazinic Acid -8.96587 1 ASN 230(3.0)
39. Sulfamethoxazole -8.94538 5 LEU 223(2.4),GLU 155(2.3)(2.4)(2.5),ASN 228(2.4)
40. Cephalothin -8.93725 5 GLU 155(2.1),HIS 219(2.4),ARG 226(2.3)(2.5)(2.8)
41. Sulfabenzamide -8.89434 4 GLU 155(2.1)(3.0),LEU 223(3.1),ASN 228(2.2)
42. Ampiroxicam -8.89059 6 AGR 226(3.2),ASN 182(2.7),GLN 220(2.3),LYS 178(2.8),SER 156(2.9),MET 180(2.9)
43. Glycyrrhizol A -8.88983 2 SER 156(2.9),LEU 224(3.5)
44. Cefprozil -8.78402 3 ASN 230(3.0),ARG 226(2.7)(2.8)
45. Sulfadicramide -8.78031 5 ASN 228(2.1),LEU 223(3.0),GLU 155(1.9)(2.5),PHE
25
116(3.3)
46. Sulfaguanole -8.77337 7 ASN 228(2.4),GLU 155(2.0)(2.1)(2.2)(2.3)(3.4),SER 156(2.9)
47. Triprolidine -8.76773 6 ARG 226(2.3)(3.0)(3.1),LEU 223(3.1),SER 156(2.8),LYS 178(3.3)
48. Glutethimide -8.7644 2 ASN 230(2.7),LYS 153(3.0)
49. Rilmazafone -8.71367 1 ASN 230(2.0)
50. Sulfamerazine -8.70442 3 ASN 230(2.5)(3.0),ASN 228(2.6)
51. Glycyrrhizol B -8.69341 4 HIS 219(2.3),ARG 226(2.9),SER 156(2.7)(2.7)
52. Diflunisal -8.69065 2 ASN 230(3.0)(2.8)
53. Cyclothiazide -8.6296 5 GLU 155(3.2)(3.0)(2.6),GLN 154(2.4),PHE 116(2.9)
54. Binifibrate -8.62216 4 ARG 226(3.0),LEU 223(2.7),MET 180(3.0),SER 156(2.7)
55. Methyclothiazide -8.62069 8 MET 180(3.0),SER 156(2.6),GLU 114(2.1)(2.3),TYR 157(2.7),HIS 177(2.8)(2.6),LYS 178(3.0)
56. Cefoxitin -8.61514 7 ARG 226(2.2)(3.1)(3.0)(2.8),LEU 223(2.9),LEU 224(3.4),GLU 155(2.9)
57. Amobarbital -8.57841 6 HIS 219(1.9)(2.1),GLU 155(1.9),LEU 223(2.5),MET 221(1.8),ARG 226(3.0)
58. Azacosterol -8.57803 1 ASN 182(2.3)
59. Isoxicam -8.56708 5 GLN 220(1.9),SER 156(2.4),MET 180(2.8)(3.0),LYS 178(3.2)
60. CiproFibrate -8.54603 2 ASN 230(2.9)(3.0)
61. Hexobarbital -8.52564 1 LYS 153(2.8)
62. Indapamide -8.51991 1 GLU 155(1.9)
63. Etaqualone -8.51263 1 LYS 178(3.3)
64. Sulfasomidine -8.47213 4 ASN 230(2.4)(3.0)(3.0),ASN 228(2.9)
65. Acimetacin -8.46989 2 ARG 226(2.4),SER 227(2.9)
66. Theofibrate -8.45029 5 MET 180(2.9),LYS 178(2.1),SER 156(2.6)(2.7),LEU 223(2.4)
67. Butoctamide -8.40024 2 GLU 155(1.9),LEU 224(2.5)
68. Chlorhexadol -8.39946 4 LEU 223(2.5)(2.8),LEU
26
224(2.6),GLU 155(3.0)
69. Acifran -8.36688 3 LYS 153(1.9)(2.4),ASN (2.7)
70. Clometocillin -8.36277 2 GLU 155(2.4),MET 180(3.0)
71. Loprazolam -8.3616 4 LYS 178(2.3),SER 156(2.8)(3.0),MET 221(3.1)
72. Lincomycin -8.35549 4 HIS 219(2.3),GLN 220(2.0),MET 180(2.9),SER 156(2.9)
73. Cinolazepam -8.35136 2 GLN 220(2.0),GLU 155(3.2)
74. Estazolam -8.34591 2 ASN 230(3.2),LYS 153(3.3)
75. Mofebutazone -8.33377 2 GLU 155(2.3),LEU 224(2.8)
76. Sulfadimethoxine -8.2973 5 ASN 230(2.7)(2.3)(3.0)(3.4),ASN 228(2.9)
77. Doxylamine -8.2898 0
78. Flunoxaprofen -8.28599 2 SER 156(2.3),MET 180(2.9)
79. Vinbarbital -8.28331 6 ARG 226(2.3)(3.0)(3.1),LEU 223(3.1),GLU 155(1.9),MET 180(2.6)
80. Cephalosporin C -8.27063 4 ARG 226(2.7)(2.8),ASN 230(2.3),LEU 223(3.0)
81. Cefmenoxime -8.26795 6 ASN 230(2.6)(3.0),ASN 228(2.5),LEU 223(2.8),LEU 224(2.8),GLU 155(3.0)
82. Propiomazine -8.24195 2 GLU 155(3.2)(2.3)
83. Ronifibrate -8.23676 2 MET 180(2.7),SER 156(2.6)
84. Atorvastatin -8.18725 1 MET 221(2.0)
85. Etofenamate -8.1836 6 MET 180(3.0),SER 156(2.6)(1.7),LEU 223(3.0),MET 221(2.8),ARG 226(2.8)
86. Cefotiam -8.17459 5 GLU 155(2.3),LEU 223(3.0)(2.8),ARG 226(3.0),HIS 219(3.1)
87. Sulfacetamide -8.16409 4 LEU 223(3.0),GLU 155(2.7)(2.4),GLN 154(3.0)
88. Lonazolac -8.15592 2 LYS 178(3.2),SER 156(2.1)
89. Apronalide -8.15258 4 LEU 223(2.8)(2.6),GLU 155(2.1)(2.1)
90. Brallobarbital -8.13998 1 MET 180(2.9)
91. Methaqualone -8.13966 2 SER 156(2.5)(2.7)
92. Hetacillin -8.1385 2 ARG 226(3.0),LEU 223(2.5)
93. Nicofibrate -8.13332 3 MET 180(3.1),ARG 226(2.9),LEU 223(2.6)
27
94. Brotizolam -8.12867 0
95. Butethal -8.12729 3 GLU 155(2.4),LYS 178(2.3),MET 180(2.5)
96. Dipyrocetyl -8.09907 4 LEU 223(2.9),LEU 224(3.0),GLU 155(3.1)(2.7)
97. Cinnamic Acid -8.08583 1 ASN 230(3.0)
98. Piperacillin -8.08384 6 ARG 226(2.9)(2.4)(2.6),MET 221(2.6),GLN 220(2.1),MET 180(2.5)
99. Niflumic Acid -8.08287 3 LYS 178(1.9),MET 180(3.0),SER 156(2.5)
100. Homofenazine -8.03812 2 ASN 182(1.9)(3.0)
101. Allobarbital -7.95645 2 MET 180(2.7),LYS 178(2.1)
102. Sulfamethomidine -7.95606 6 ARG 226(2.7)(3.0),GLU 155(2.0)(3.5)(2.8)(3.0)
103. Sulbenicillin -7.92462 3 SER 156(3.0)(2.1),GLU 155(3.5)
104. Cefazolin -7.92151 1 LYS 153(2.8)
105. Xipamide -7.91964 ARG 226(2.3)(3.0)(3.1),LEU 223(3.1),GLU 155(2.7),SER 156(3.2)
106. Sulfasomizole -7.89342 4 GLU 155(2.2)(2.2)(2.9),LEU 223(2.5)
107. Nealbarbital -7.88849 1 SER 156(2.7)
108. Sulfametrole -7.88622 4 GLU 155(2.8)(3.3),MET 221(3.5),LEU 223(3.0)
109. Butallylonal -7.8725 1 SER 156(2.8)
110. Secobarbital -7.8635 4 MET 221(2.0),ARG 226(3.0),LEU 223(2.6),GLU 155(2.8)
111. Clopamide -7.81042 5 GLU 155(2.3),SER 156(2.9),ARG 226(3.0),MET 221(3.2),LEU 223(2.2)
112. Niaprazine -7.7984 2 SER 156(3.1)(2.8)
113. Metolazone -7.77043 1 GLY 185(3.6)
114. Eugenol -7.75931 2 LEU 224(3.0),LEU 223(2.2)
115. Enallylpropymal -7.74731 1 MET 180(2.9)
116. Sulfachrysoidine -7.74296 8 MET 180(3.0),SER 156(2.1),LYS 178(2.9)(2.9),GLU 114(2.8)(2.2)(3.4)(2.4)
117. Bromosaligenin -7.73911 2 LEU 224(3.0),GLU 155(2.1)
118. Isoglycyrol -7.73752 3 LEU 223(2.0)(2.9),ARG 226(3.0)
28
119. Holomycin -7.73712 2 HIS 177(2.6),LYS 178(2.3)
120. Pivampicillin -7.73595 6 LEU 223(2.5),GLU 155(1.7)(2.0),ARG 226(3.4),HIS 219(2.5),MET 221(2.4)
121. Zolpidem -7.6811 6 SER 156(2.7),LYS 178(3.0), ARG 226(2.3)(3.0)(3.1),LEU 223(3.1)
122. Sulfadoxine -7.67583 5 HIS 219(3.2),ARG 226(2.9),MET 221(2.9),LEU 223(2.3),LYS 153(2.3)
123. Ribostamycin -7.66203 6 HIS 219(2.1),GLU 155(2.2)(2.0),SER 156(2.9)(2.5),LEU 224(3.0)
124. Butabarbital -7.64791 6 MET 180(2.6),LYS 178(2.6),SER 156(2.2)(2.9),GLU 155(2.3)(2.0)
125. Cefditoren -7.62905 2 MET 180(2.2),SER 156(2.2)
126. Novonal -7.58444 2 PHE 116(2.4),GLU 155(2.1)
127. Ceftezole -7.57994 6 GLN 220(3.4),MET 180(2.3),LYS 178(2.7),SER 156(2.4)(2.7)(3.1)
128. Carbubarb -7.54777 5 GLU 155(2.3),SER 156(2.4)(2.1),GLU 114(1.9),LYS 178(2.5)
129. Tetroxoprim -7.54307 3 ARG 226(2.9),LEU 223(2.9)(3.0)
130. Triamterene -7.51217 0
131. Sulfaguanidine -7.46617 2 GLU 155(2.5)(2.8)
132. Grepafloxacin -7.4515 3 ASN 230(3.0)(2.4),GLU 155(1.8)
133. Clofibrate -7.42224 3 ASN 228(3.0),ASN 230(3.0)(2.5)
134. Clopirac -7.37794 3 LYS 178(2.4),SER 156(1.9)(2.7)
135. Methicillin -7.36738 3 ASN 230(2.1),LYS 153(2.6),LEU 223(2.4)
136. Amphenidone -7.35826 3 HIS 219(2.6),LEU 224(2.6),LEU 223(2.7)
137. Isoniazid -7.33465 2 ASN 228(2.4),ASN 230(3.6)
138. Cefotaxime -7.32019 5 GLU 155(2.2)(3.5),SER 156(2.8)(2.1),MET 180(3.0)
139. Allyl Disulfide -7.31593 0
140. Nalidixic Acid -7.28493 2 SER 156(2.7),LEU 224(3.0)
141. Ceftraxone -7.13541 4 HIS 219(2.3),ASN 182(1.9),MET 180(3.5),LYS 178(2.1)
142. Pirozadil -7.13018 3 ARG 226(2.4)(3.0),LEU 223(2.3)
143. Hydracarbazine -7.12728 4 MET 180(3.0),SER 156(2.4)(2.9),HIS 219(2.6)
29
144. Methazolamide -7.0841 5 ARG 226,MET 221(2.6),MET 180(3.1)(3.0),SER 156(2.4)
145. Cinoxacin -7.05223 3 ARG 226(2.8)(2.8),SER 156(2.2)
146. Meropenem -7.04503 1 LYS 178(2.1)
147. Clomethiazole -7.00835 0
148. Epirizole -6.99144 3 LYS 178(2.9),MET 180(3.0),SER 156(2.9)
149. Tosufloxacin -6.98631 3 ASN 182(2.0),ARG 226(2.8),LEU 223(2.9)
150. Temafloxacin -6.98207 1 SER 156(3.5)
151. Meparfynol -6.93231 2 ASN 230(3.0),ASN 228(2.1)
152. Pyrithyldione -6.89853 3 MET 180(3.0),SER 156(3.5),LYS 178(1.9)
153. Trimethoprim -6.87919 4 ARG 226(2.3)(3.0)(3.0),LEU 223(3.0)
154. Nitrofurantoin -6.84307 3 GLN 220(2.3),ARG 226(3.0),LEU 223(3.0)
155. Trovafloxacin -6.81609 5 MET 180(2.5), ARG 226(2.3)(3.0)(3.1),LEU 223(3.1)
156. Narcobarbital -6.79219 2 LYS 178(2.3),MET 180(3.4)
157. Hexedine -6.78224 0
158. Pipemidic Acid -6.77423 3 SER 156(2.9),LYS 153(3.4),LEU 224(3.0)
159. Tripelennamine -6.7399 4 ARG 226(2.3)(3.0)(3.1),LEU 223(3.1)
160. Hexanal -6.69026 2 GLU 155(2.7),GLN 154(3.0)
161. Norfloxacin -6.68088 2 PHE 116(2.8),GLN 117(2.6)
162. Sparfloxacin -6.59288 3 ARG 226(2.7)(3.0),LEU 223(2.7)
163. Fleroxacin -6.5803 1 GLU 155(2.7)
164. Sulfonmethane -6.55214 6 MET 180(3.0),LYS 178(2.9),SER 156(2.6)(2.9),SER 156(3.1)(3.2)
165. Enoxacin -6.46284 4 MET 180(3.0)(2.6),LYS 178(2.1),SER 156(2.9)
166. Hexane -6.38915 0
167. Paraldehyde -6.3786 1 GLU 155(2.5)
168. Isosorbide -6.34751 7 ARG 226(2.3)(3.1),GLU 155(2.5)(3.3),LEU 224(2.7),LEU 223(3.0)(2.8)
169. Theobromine -6.29539 2 SER 156(2.3),LYS 178(2.1)
170. Piperidine -6.17934 1 GLU 114(1.8)
30
171. Acipimox -5.94946 2 AGR 226(3.0),LEU 223(2.9)
172. Acetone -5.82875 2 LEU 224(2.9),LEU 223(2.8)
173. Ethanol -5.44075 1 HIS 177(2.4)
174. Methenamine -5.23975 1 SER 156(2.7)
Figure . 7a Diathymosulfone - The top most ligand compound showing interaction with its protein
Leukocidin D
31
Figure. 7b Myophine compound showing interaction with its protein Leukocidin D
Figure. 7c Andrimid compound showing interaction with its protein Leukocidin D
32
Figure. 7d Beclobrate compound showing interaction with its protein Leukocidin D
Figure. 7e Probucol compound showing interaction with its protein Leukocidin D
33
CONCLUSION Using the blast search, the A covalent S-F heterodimer of staphylococcal gamma-
hemolysin Chain B was obtained as a suitable template with the Query coverage of
90%, Maximum identity of 77% This proves that 2qk7 is a suitable template for
Leukocidin D.
Eight structures were generated through four different servers EsyPred3D, Swiss
model, Modeller and Phyre2. Out of the structures generated swiss model was found
as suitable model based upon the Ramachandran plot and visualization of the
superimposition between target and the template. The RMSD deviation for this
structure is 0.077Å which is also low compared to other predicted models.
The stereochemistry of the constructed structure of Leukocidin D was subjected to
energy minimization and the stereo chemical quality of the predicted structure was
assessed. The Ramachandran plot for the model showed 82.7% of the residues in the
core region, 11.7% residues in the additionally allowed regions, 0.8% of residues in
the generously allowed region and 0.4%. in the disallowed region. The overall verify
3D quality was 98.66% and the ERRAT value was 83.394. Further the energy of the
protein obtained by ProSA was -5.06 which shows Z score of -5.06.
Through the results from Q site finder Possible ligand binding sites are predicted with
the residue name and numbers (ARG 226,ASN 228,GLN 154,GLU 155,HIS 219,ILE
179,LEU 223,LEU 224,LYS 153,LYS 178 MET 180,PHE 116,PRO 222,SER
156,TYR 157).
Using Argus Lab, ligand compounds were then taken one by one for interacting with
Leukocidin D. After ligand file preparation docking energy was calculated by dock a
ligand. Docking energy were documented during docking. The interaction between
the ligand and the protein were then viewed in PyMol viewer. Diathymosulfone and
Myophine are the two top most predicted ligands with high docking energy.
Andrimid, Beclobrate, Probucol are the other three best compounds next to the top
two compounds.
34
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