Homology Modeling, Simulation and Docking
Studies of Tau-Protein Kinase
A. Ranganadha Reddy1*, T.C. Venkateswarulu1,
D. John Babu1, N. Shyamala Devi2
1School
of Biotechnology, Vignan University, Vadlamudi, Guntur, 522213, India
2Department
of Biotechnology, Sree Vidyanikethan Engg. College, Tirupathi, 517102, India
*Corresponding Author E-mail: rangaaluri@gmail.com
Alzheimer’s disease is a brain disorder
characterized by the most common form of dementia associated with plaques and
tangles in the brain. The most commonly recognized symptoms are memory loss,
such as difficulty in remembering and occasional problems with remembering
certain things and confusion. These symptoms are signs of a failing brain
cells. The brain has 100 billion nerve cells (neurons).Each nerve cell
communicates with many others to form networks. In Alzheimer’s disease,
increasing numbers of brain cells deteriorate as in other types of
dementia.Tau-protein kinase (EC 2.7.11.26)is an enzyme that catalyzes the
transferring of a phosphate group to the side chain oxygen atom of serine or
threonine residues.
Tau-protein is an important protein of the
central nervous system that stabilizes microtubules and neurons. Abundant
neurofibrillary lesions made of hyperphosphorylated Tau-protein constitute one
of the defining neuropath logical features of Alzheimer’s disease. Tau-proteins
interact with tubulin to stabilize microtubules. Pathogenesis of Alzheimer’s
disease includes hyperphosphorylation of the tau-protein which results in the
self-assembly of tangles of paired helical fragments and straight filaments.
Defective Tau-protein causes the dementia leading to Alzheimer’s disease.
Considering the importance and lack of
structural information, we modeled Tau protein with homology modeling and
performed docking studies with L-Glutamic acid, Memantine, Tacrine, Ropinirol
ligand. The minimized modeled structure has shown good structure similarity
with template and show high binding association energy with ligand.
KEYWORDS: Alzheimer, Tau protein, Homology modeling, Simulation,
Docking.
INTRODUCTION:
Alzheimer's disease (AD) is a
neurodegenerative disease of the central nervous system .Alzheimer's disease (AD), also called Senile Dementia of the
Alzheimer Type (SDAT) or simply Alzheimer's, is the most prevalent form of
dementia. Alzheimer's
disease is incurable,
neuro degenerative, and terminal disease and was first described by German
psychiatrist and neuropathologist Alois Alzheimer in 1906 and was named after
him.[1] It is generally diagnosed, in people aged 65 years, [2]
although the less-prevalent early-onset Alzheimer's can occur much earlier.
According to the statistics of 2006, there were 26.6 million sufferers
worldwide. The prevalence of Alzheimer's is expected to reach approximately 107
million people by 2050.[3]
Even though the course of Alzheimer's disease is unique for
every individual, there are many common symptoms.[4] The earliest
observable symptoms are often mistakenly thought to be 'age-related' concerns,
or manifestations of stress.[5] In the preliminary stages, patient
loses his memory and feel difficulty in remembering recently learned facts.
When a doctor or physician has been notified, and AD is suspected, the diagnosis
is usually concerned with behavioural assessments and cognitive tests, which
are often followed by a brain scan if available.[6]
As the disease advances, symptoms include irritability,
confusion, mood swings, aggression, long-term memory loss, language breakdown,
and the general withdrawal of the sufferer as their senses get declined.[5][7]
Gradually, bodily functions cease, ultimately leading to death.[8]
Individual prognosis becomes difficult to assess, as the duration of the
disease varies. Apart from major symptoms, two symptoms are playing a vital
role which is associated with Alzheimer’s disease. The primary symptom of
Alzheimer’s disease is cognitive type of symptoms. They are Amnesia (short term
and long term memory loss), aphasia, agnosia and apraxia [9]. The
secondary symptom of Alzheimer’s disease is psychiatric type of symptoms. They
are personality changes, depression, hallucinations and delusions. AD develops
for an indefinite period of time before becoming fully apparent, and it can
remain undiagnosed for years. The average (mean) life expectancy following
diagnosis of AD is approximately seven years.[10] After diagnosis
Less than three percent of individuals live more than fourteen years.[11]
The reasons for the cause and progression of Alzheimer's
disease are not well understood. Earlier studies indicate that the disease is
associated with tangles and plaques in the brain.[12] Currently used
treatments give a small symptomatic benefit; no treatments to delay or halt or
stop the progression of the disease are not yet available. As of 2008, more
than 500 clinical trials have been carried out for identification of a suitable
treatment for AD, but it is unknown if any of the tested intervention
strategies will show promising results.[13] A number of
non-invasive, life-style habits have been suggested for the prevention of
Alzheimer's disease, but there is a lack of adequate evidence for a link
between these recommendations and reduced degeneration. Mental stimulation,
exercise, and a balanced diet are suggested, as both a possible prevention and
a sensible way of managing the disease.[14]
Management of AD patients is essential because it cannot be
cured and is degenerative. Main caregiver role is often played by the spouse or
a close relative.[15] Alzheimer's disease is well known for placing
a great burden on caregivers; the pressures can be wide-ranging, involving
psychological, social, physical, and economic elements of the caregiver's life.[16][17[18]
AD is one of the most costly diseases to society in developed countries.[19][20]. A tau protein
is a protein found in neurons, primarily
in the central nervous system. Tau
proteins are proteins
that stabilize microtubules. Phosphorylation of tau is regulated by a
host of kinases. For example, PKN, a serine/threonine
kinase. When PKN is activated, it phosphorylates tau, resulting in
disruption of microtubule organization.[21] Hyperphosphorylation
of the tau protein (tau inclusions), however, can result in the self-assembly of tangles of
paired helical filaments and straight filaments, which are involved in the
pathogenesis of Alzheimer's
disease and other Tauopathies.[22]
Computational Tools
All calculations were carried out in
Maestro v9.2 installed in Cadd-WS3 machine under 64-bit centos operating system
placed in CADD department, Institute of Life Sciences. The machine was built up
with:
A) 4 cores and 8 processers with
Intel Xenon CPU E5620 @ 2.40GHZ
B) 16 GB RAM
C) NVidia Qudvo FX3800 Graphical
Process Unit (GPU)
D) The PROCHECK analysis provides
an idea of the stereo chemical quality of all protein chains in a given PDB
structure. They highlight regions of the proteins which appear to have unusual
geometry and provide an overall assessment of the structure as a whole.
E) Other Servers
1) Primary sequence of the
TAU-PROTEINKINASE was retrieved from Swiss Prot (accession number Q5TCY1) from the ExPASy (Expert
Protein Analysis System) proteomics serves of the Swiss Institute of
Bioinformatics.
2) Homology search for
TAU-PROTEINKINASE was carried out using BLAST software.
F) The crystal structure for
TAU-PROTEINKINASE (PDB ID: 1CKI) was obtained from PDB database RCSB.
G)
BLAST:
Web based online tool searches for high scoring sequence alignments between the query sequence and
sequences in the database using a heuristic approach that approximates the Smith-Waterman algorithm. Protein-protein BLAST (blastp):
This program, given a protein query, returns the most similar protein sequences
from the protein database that the user specifies. http://blast.ncbi.nlm.nih.gov
SOPMA:
The Self-Optimized
Prediction method With Alignment is a tool to predict the secondary structure
of a protein. Based on the query (primary sequence of a protein), SOPMA will
predict its secondary structure. http://www.expasy.org
Web based servers
RAPPER is an
ab initio conformational search algorithm for restraint-based protein
modelling. It has been used for all-atom loop modelling (DePristo et al., de
Bakker et al.), whole protein modelling under limited restraints (DePristo et
al.), comparative modelling (de Bakker et al., in preparation), ab initio
structure prediction, structure validation (Lovell et al.), and experimental
structure determination with X-ray and nuclear magnetic resonance spectroscopy
(DePristo et al.).Web interfaces are available on this website for Ramachandran plot analysis.
CASTP (computed atlas of surface
topography of proteins)
Binding sites and active sites of proteins
and DNAs are often associated with structural pockets and cavities. CASTp is updated daily and can be
accessed at http://cast.engr.uic.edu.
Software Used:
BIOEDIT:
BioEdit is a
mouse-driven, easy-to-use sequence alignment editor and sequence analysis
program, In-color alignment and editing with separate nucleic acid and amino
acid color tables and full control over background colors.
MOLSOFT:
Molsoft is a La Jolla; California based
company that is a primary source of new breakthrough technologies in molecular
graphics and visualization, molecular modeling, docking and virtual screening,
computational biology and chemistry.
Figure
1: The four main steps of comparative protein
structure modeling: template selection, target–template alignment, model
building and model quality evaluation.
Discovery Studio:
Discovery
Studio (DS) is a complete modeling and simulations environment for Life
Scientists. Provides tools for visualization, modeling, simulations, docking,
pharmacophore analysis and much more.
Argus Lab:
Argus Lab is a molecular modeling
program that runs on Windows 98, NT, and 2000. ArgusLab consists of a user
interface that supports OpenGL graphics display of molecule structures and runs
quantum mechanical calculations using the Argus compute server. The Argus
compute server is constructed using the Microsoft Component Object Model
GOLD (Genetic optimization for ligand docking):
It is a program for calculating
the docking modes of small molecules in protein binding sites and is provided
as part of the GOLD Suite, a package of programs for structure
visualization and manipulation (Hermes), for protein-ligand docking (GOLD) and for post-processing
(Goldmine) and visualization of docking results.
NAMD/VMD
NAMD (NAnoscale Molecular
Dynamics)1is a free-of-charge molecular dynamics simulation package written
using the Charm++ parallel programming model, noted for its parallel efficiency
and often used to simulate large systems (millions of atoms). VMD is primarily
developed as a tool for viewing and analyzing the results of molecular dynamics
simulations, but it also includes tools for working with volumetric data,
sequence data, and arbitrary graphics objects.
Procedure
To build a homology model of Tau-protein
kinase (Tau-tubulin kinase) and to carry out the molecular dynamics simulation
studies by VMD/NAMD. Later on docking studies were carried out using Argus Lab
with the stable structure and finally performed flexible docking studies in GOLD.
A typical PDB structure consists
of heavy atoms, waters, cofactors, metal ions and can be multimeric. The
structure generally has no information on bond orders, topologies, or formal
atomic charges. So, 1CKI (from the PDB) must be prepared by using
protein preparation wizard (PrepWiz) of Schrödinger software. Protein
preparation ensures that the 1CKI protein structure was properly
assigned with bond orders and correct number of hydrogens to make the structure
compatible with the OPLS (Optimized Potential for Liquid Simulations)
forcefields [23]. In the process of building the homology
model Accelrys Discovery studio is used.
Homology modeling
The
steps to creating a homology model are as follows:
Ø Identify homologous proteins and determine
the extent of their sequence similarity with one another and the unknown.
Ø Align the sequences.
Ø Identify structurally conserved and
structurally variable regions s
Ø Generate coordinates for core (structurally
conserved) residues of the unknown structure from those of the known
structure(s).
Ø Generate conformations for the loops (structurally
variable) in the unknown structure.
Ø Build the side-chain conformations.
Ø Refine and evaluate the unknown structure.
Figure-1 represents the four main steps
of homology modeling of protein i.e. template
selection, target–template alignment, model building and model quality
evaluation [24].
Structure Minimization
For energy minimization of the
generated models, MacroModel module was utilized. The default parameters were
used i.e. OPLS_2005 was selected as the forcefield, convergence threshold was
set to 0.05 and 15000 iterations were specified. The job got completed
immediately after the convergence had reached.
Molecular Dynamics
MacroModel dynamics was performed to the
structure resulted from minimization. In the Molecular Dynamics all hydrogen atoms,
ions, and water molecules were first subjected to 500 steps of energy
minimization by steepest descent algorithm to remove undesired close vander
Waals contacts. The temperature of the system was kept 323K.
Figure-2: The above graph is
showing amino acid composition of Query sequence, the amino acid LEU is present
highest percentage compared to other residues and the molecular weight of the
protein is 10.61.
Time step value altered to 1.0 fs used in
the integration of the equations of motion during the simulation. Equilibration
time kept default value 1.0 ps used to determine the length of the settling
down period at the start of the simulation. Number of structures to sample the
receptor protein structure (Tau protein kinase) was given 100. The convergence
of simulation was analyzed in terms of the potential energy gradient and the
convergence threshold is 0.05 is a default value. Force field OPLS_2005 was
used with a constant dielectric of 1.0. Solvent was used to run the protein
system in solvent phase. The energy equilibrated molecular structure was
computed upon completion of the Molecular Dynamics simulation, and the averaged
structure was energy re-minimized to eliminate errors associated out of
averaging exercise for obtaining the final model [25].
Docking
Molecular
docking is a key tool in structural molecular biology and computer-assisted
drug design. The goal of ligand—protein docking is to predict the predominant
binding mode(s) of a ligand with a protein of known three-dimensional structure.Docking is a method which predicts
the preferred orientation of one molecule to a second when bound to each other to form a stable complex molecule. Knowledge of the preferred
orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for
example scoring
functions.
RESULTS AND DISCUSSION:
Sequence Retrieval and Data Collection
The details of the protein Tau protein
kinase like amino acid lengths of N-and C-terminus, helix lengths etc, were obtained
from Protein knowledgebase UniProtKB/Swiss-Prot database with accession number
Q5TCY1 (Tau protein kinase). The details of sequence annotation are
shown below.
Query sequence of Tau-tubulin kinase
>sp|Q5TCY1|TTBK1_HUMAN Tau-tubulin kinase 1 OS=Homo sapiens GN=TTBK1 PE=1 SV=2 WKVLKKIGGGGFGEIYEAMDLLTRENVALKVESAQQPKQVLKMEVAVLKKLQGKDHVCRFIGCGRNEKFNYVVMQLQGRNLADLRRSQPRGTFTLSTTLRLGKQILESIEAIHSVGFLHRDIKPSNFAMGRLPSTYRKCYMLDFGLARQYTNTTGDVRPPRNVAGFRGTVRYASVNAHKNREMGRHDDLWSLFYMLVEFAVGQLPWRKIKDKEQVGMIKEKYEHRMLLKHMPSEFHLFLDHIASLDYFTKPDYQLIMSVFENSM
Sequence Analysis by Bioedit
Bioedit analysis of query protein Q5TCY1 shown in Figure-2 and Figure-3 indicates that the amino acid Leucine is present highest percentage compared to other residues and the molecular weight of the protein is 10.61.Similarly Helical wheel diagram shown in Figure-4 obtained using Genetic Computer group shows the relative positions of amino acids, hydrophobic residues located in core region. Kyte and doolittile program shown in Figure-5 is used to identify hydrophobic regions, the plot reliably predict 4 hydrophobic regions above the slidding window, which are located in the three dimensional structure at amino acid positions 40-50,100-119,190-210238-245.Secondary structure analysis of Tau-protein kinase Q5TCY1 shown in Figure-6 was carried out using SOPMA and the result showed the percentage of Alpha helix (Hh) is 40.91%, percentage of 310 helix (Gg) is 0.00% , percentage of Pi helix (Ii) is 0.00% , percentage of Beta bridge (Bb) is 0.00% , percentage of Extended strand (Ee) is 15.53% , percentage of Beta turn (Tt) is 6.06% , percentage of Bend region (Ss) is 0.00% , percentage of Random coil (Cc) is 37.50% percentage of Ambigous states is 0.00% and Other states is 0.00%.
Amino acid composition
Figure-3: The above graph is showing amino
acid composition of Query sequence, the amino acid Leu is present highest
percentage compared to other residues.
Figure-4: Wheel plot of the Tau-protein
kinase. the plot Shown obtained using Genetic Computer group HELICAL WHEEL
PROGRAM, the diagram shows the relative positions of amino acids, hydrophobic
residues located in core region
Figure-5: Hydrophobicity plot of query
sequence, Kyte and doolittile program was used to identify hydrophobic regions,
the plot reliably predict 4 hydrophobic regions above the slidding window, which
are located in the three dimensional structure at amino acid positions
40-50,100-119,190-210238-245.
WKVLKKIGGGGFGEIYEAMDLLTRENVALKVESAQQPKQVLKMEVAVLKKLQGKDHVCRFIGCGRNEKFN
eeeeeeecttccceeeehhhhccccheeeeecccccchhhhhhhhhhhhhhttccccceeeeccccttce
YVVMQLQGRNLADLRRSQPRGTFTLSTTLRLGKQILESIEAIHSVGFLHRDIKPSNFAMGRLPSTYRKCY
eeeehhhccchhhhhhhccccccchhhhhhhhhhhhhhhhhhhhtteecccccccceeeeccccccceee
MLDFGLARQYTNTTGDVRPPRNVAGFRGTVRYASVNAHKNREMGRHDDLWSLFYMLVEFAVGQLPWRKIK
eeettcchhecccttccccccccccccccceeehhhhhttccccchhhhhhhhhhhhhhhttcccccccc
DKEQVGMIKEKYEHRMLLKHMPSEFHLFLDHIASLDYFTKPDYQLIMSVFENSM
Ccchhhhhhhhcchhhhhhcccchhhhhhhhhhhhcccccccchhhhhhhhhhh
Sequence length : 264
SOPMA :
Alpha helix (Hh) : 108 is 40.91%
310 helix (Gg) : 0 is 0.00%
Pi helix (Ii) : 0 is 0.00%
Beta bridge (Bb) : 0 is 0.00%
Extended strand (Ee) : 41 is 15.53%
Beta turn (Tt) : 16 is 6.06%
Bend region (Ss) : 0 is 0.00%
Random coil (Cc) : 99 is 37.50%
Ambigous states (?) : 0 is 0.00%
Other states : 0 is 0.00%
Parameters :
Window width : 17
Similarity threshold : 8
Number of states : 4
Figure-6: Secondary structure analysis of Tau-protein kinase
Figure-7: BLAST result for Tau-protein kinase
The Basic Local Alignment Search Tool
(BLAST) finds regions of local similarity between protein or nucleotide
sequences. The program compares nucleotide or protein sequences to sequence in
a database and calculates the statistical significance of the matches. The
target sequence i.e., Tau-protein
kinase (UniProt ID:
Q5TCY1) was searched against the protein database by using
BLAST tool. From the BLAST results as shown in Figure-7 I observed that
three proteins (PDB IDs: 1CKI, 1CKJ, 2CK7) are showing the maximum identity
with the target sequence. Among the three proteins obtained, 1CKI
selected for further proceedings.
Three-Dimensional Structure Prediction
by MOLSOFT
Three dimensitional structure of
Tau-protein kinase (Q5TCY1) as shown in Figure-8 was predicted by using the
tool MOLSOFT ICM by taking 1CKI as template which was obtained through BLAST
results by taking Q5TCY1 as query sequence and performing Protein BLAST against
the protein sequence database.Similarly three dimensitional structure of
Tau-protein kinase (Q5TCY1) as shown in Figure-9 was predicted by using
Discovery studio taking 1CKI_A and 1CKI_B chains as Templates which are homologus
to tau-protein kinase (query).Alignment between the input query sequence
Q5TCY1and the templates 1CKI_A and 1CKI_B was carried out using Discovery
Studio is shown in Figure-10.
Figure-8: Tau-protein kinase structure was
predicted by Molsoft ICM, by taking template 1CKI_A
Three –Dimensional Structure of Tau-Protein
Kinase Predicted by Discovery Studio
Figure-9: Templates 1CKI_A and 1CKI_B
chains are homologus to tau-protein kinase (query).
Input Sequence Alignment:
Figure-10: Aligning the query sequence with
template
Build Homology Models Description
Homology Modeling was carried out using
Accelrys Discovery Studio with query sequence (Tau-protein kinase Q5TCY1) and
1CKI_A and 1CKI_B as templates the results of which are shown in Figure-11, Figure-12
and Figure-13 respectively. Finally we got a model whose RMS deviation is 0.75
after Superimposition of Tau-Protein kinase structure with Templates 1CKI_A and
1CKI_B.
Figure-11: Secondary structure
visualization of Tau-Protein kinase
Three-Dimensional Structre of Tau-Protein
Kinase
Figure-12: Superimposition Query
structure with templates
Figure-13: Superimposition of
Tau-Protein kinase structure with Template, the RMS deviation is 0.75.
Figure-14: Ramachandran plot
analysis of the generated model
The validation of the final model was
carried out using Ramachandran plot computed with PROCHECK, program by checking
the detailed residue-by-residue stereo-chemical quality of a protein structure.
The PROCHECK is used for stereochemical assessment of the model. The criteria
for analysis of stereochemistry of the model includes,
1) Main chain conformation in
acceptable regions of the Ramachandran plot.
2) Planar peptide bonds.
3) Side chain conformations that
correspond to those in rotamer library.
4) Hydrogen bonding of polar atoms
if they are buried.
5) No bad atom-atom contacts.
6) No holes inside the structure.
Ramachandran Plot
A Ramachandran plot (also known as a Ramachandran map or a Ramachandran diagram or a [Φ,Ψ] plot), developed by Gopalasamudram Narayana Ramachandran and Viswanathan Sasisekharan is a way to visualize dihedral angles Ψ and Φ of amino acid residues in protein structure. It shows the possible conformations of Φ and Ψ angles for a polypeptide. Hence, Ramachandran plot is a useful way of assessing the stereo chemical quality of a protein structure. From the main Ramachandran plot developed by PROCHECK our Tau kinase protein had 247 (94.3%) residues in favoured region against (~98.0% expected), 10 (3.8%) residues in allowed region against (~2.0% expected) and 5 (1.9% residues in outlier region as shown in Figure-14.
Molecular Dynamics Simulation Studies by
VMD/NAMD:
The modeled structure was
validated stereo chemically after the energy minimization process by VMD/NAMD
and modeled structure was shown very less RMSD with its template .Structure
having least energy with low Rmsd value obtained through NAMD is shown in
Figure-15 and Figure-16 below. Similarly structure having least energy with low
Rmsd value Obtained through VMD is shown in Figure-17 and Figure-18.
Figure-15: Molecular File Browser
Figure-16: Structure having least energy
with low Rmsd which was obtained by NAMD in water molecule (TIP)
Figure-17: Command Window of VMD
Figure- 18:Graphical Representation of RMSD
value of Tau-Protein Kinase
Identificatin of active sites of
proteins and DNAs are often associated with structural pockets and cavities
were carried out by using CASTp. CASTp is updated daily and can be accessed at
http://cast.engr.uic.edu. CASTp predicted different active site Pockets based
on area and volume we have selected the best pocket as 17 is shown in
Figure-19.
Figure-19: CASTp predicted different active
site Pockets based on area and volume we have selected the best pocket as 17
Docking Studies
The stable structure is used for docking studies with
L-Glutamic acid, Mematnine, Tacrine, and Ropinirol in ARGUS LAB as shown in
figure-20 and figure-21.The interactions between the modeled stable Tau-protein
structure and ligand molecules and their energy in stable conformations are
shown in Table-1. Among all of them Mematnine got least energy (-51.31263) and
high affinity.
Argus Lab Results
Figure-20: Docking of stable structure with
Ligand molecules
Table: 1 List of Drug molecules and their
energy values in K/Cal
Figure-21: Best ligand Pose energy is
-9.0571
GOLD Results:
Further studies we have taken similar compounds of Memantine
( 40compounds) and performed flexible docking studies in GOLD, there we got
better drug molecule (5R,7R)-3-propyladamantan-1-amine
it was shown strong hydrogen bonding interactions with Glu14,Val 31,Leu 29 as shown in Figure-22.
Figure-22: The drug molecule was
interacting with active site residues Hbonds interaction formed between Glu14,
Val 31, and Leu29
CONCLUSION:
Present study we have modeled
Tau-proteinkinase which has key functional role in causing the Alzheimer disease. We selected the
template by using BLAST tool and the template shown high similarity with its
template 1CKA shows high accuracy. The modeled structure was validated stereo
chemically after the energy minimization process by NAMD and modeled structure
was shown very less RMSD with its template. The stable structure is used for
docking studies with L-Glutamic acid, Mematnine, Tacrine, and Ropinirol in
ARGUS LAB. Among all of them Mematnine got least energy (-51.31263) and high
affinity. Further studies we have taken similar compounds of Memantine (40compounds)
and performed flexible docking studies in GOLD, there we got better drug
molecule (5R,7R)-3-propyladamantan-1-amine
it was shown strong hydrogen bonding interactions with Glu14,Val 31,Leu 29 .
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