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Structural Characterization, Homology Modeling and Docking Studies of ARG674 Mutation in MyH8 Gene Associated with Trismus-Pseudocampto-dactyly Syndrome

Munazzah Tasleem, Romana Ishrat, Asimul Islam, Faizan Ahmad and Md. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 10025, India

Abstract: Trismus-pseudocamptodactyly (TPS) syndrome is a musculoskeletal disorder, caused by mutation in the peri-natal MyH8 gene, leading to the incomplete opening of mouth and camptodactyly of fingers upon dorsiflexion of the wrist. MyH8 gene is relevant for muscle regulation, and it plays a significant role in muscle motor function, the hydrolysis of ATP, that is essential for the production of force for muscle movement. To understand the structural basis of TPS, we utilize a large number of software packages to estimate how the Arg674Gln mutation would affect the structure and stabil-ity of MyH8 gene product. The motor domain of MYH8 was modeled in order to know what are the interactions altered in the mutant protein. Further, we docked the ATP in the nucleotide binding region of both wild type and Arg674Gln mutant to understand the mechanism of action. Our strategy may be helpful for understanding the mechanism of regulation of muscle movements and the role of Arg674Gln mutation in TPS.

Keywords: Docking, Homology modeling, MyH8 gene, Myosin, Protein-protein interaction and Trismus-pseudocamptodactyly syndrome.

INTRODUCTION

Musculoskeletal diseases became a major public health problem. Several studies have been conducted to establish the prevalence of the musculoskeletal diseases [1-6]. It was found that MyH8 gene is also associated with different mus-culoskeletal disorders, including trismus-pseuodocamp-todatcyly (TPS) syndrome [7-9], an autosomal dominant disorder, also known as distal arthrogryposis (DA) type 7 [10] and Dutch-Kentucky syndrome [9].

TPS is manifested by the incomplete opening of the mouth known as trismus and camptodactyly of the fingers that appears only upon dorsiflexion of the wrist, known as pseudocamptodactyly [9]. MyH8 is expressed only in the fetal developmental period during muscle regeneration [11], primarily in the skeletal muscles of the limbs and to a more limited extent in the muscles of the face. Therefore, the con-tractures that appear in TPS are prenatally, confined to the muscles of the limb and face, and are non-progressive [9].

TPS has been reported to be caused by a single missense mutation, c.2021G>A (p.Arg674Gln) in perinatal myosin heavy chain gene [12]. Arg674 is evolutionary conserved residue, localized in the actin-binding domain of the perina-tal myosin head. This residue is also close to the ATP-binding site [13]. We have been working on the structural based drug designing for that we first need the modeled structure of protein [14-17]. Moreover, sequence analyses have provided a better insight into the function of particular protein [18, 19]. Hence, we have performed the sequence

*Address correspondence to this author at the Centre for Interdisciplinary Research In Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 10025, India; Tel/Fax: +91-11-2698-3409; E-mail: mihassan@jmi.ac.in

analysis of MyH8 followed by homology modeling, to iden-tify its functionally important residues. We built the model of both wild type and Arg674Gln mutant. Structures were validated for their quality and optimized models were used for structure analysis and comparison. Protein ligand interac-tion analysis is essential to understand the precise function of a particular residues in the binding, and hence function [20-22]. Here, we docked ATP at the nucleotide binding domain of both wild type and Arg674Gln mutant MyH8 to know the functional significance of Arg674Gln mutation.

MATERIALS AND METHODS

Sequence and Pathways Analysis

The complete sequence of MyH8, containing 1937 amino acid residues, was retrieved from UniprotKB database (http://www.uniprot.org). Identification of motifs and do-main architecture was performed using computational tools MOTIFsearch (http://www.genome.jp/tools/motif/), Pfam (http://pfam.sanger.ac.uk/), and SMART (http://smart.embl-heidelberg.de/) [23]. We selected sequences of MyH8 from 28 genus, namely, Chinchilla lanigera, Gallus gallus, Microtus ochrogaster, Cavia porcellus, Heterocephalus glaber, Mustela putorius furo, Condylura cristata, Sorex araneus, Dasypus novemcinctus, Ceratotherium simum simum, Odobenus ros-marus divergens, Trichechus manatus latirostris, Orcinus orca, Nomascus leucogenys, Saimiri boliviensis boliviensis, Papio anubis, Pongo abelii, Callithrix jacchus, Rattus nor-vegicus, Cricetulus griseus, Macaca fascicularis from the UniProtKB/Swiss-Prot that provides a high quality annotated and non-redundant protein sequences. Multiple sequence alignments of MyH8 protein with numerous genus were gen-erated using Discovery Studio3.5 and ClustalW2 [24].

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It has been reported that functionally interdependent pro-teins are more likely to be connected in molecular networks. Interaction of MyH8 with other proteins for its proper func-tioning was predicted using online server STRING (http://string-db.org/). Pathways analysis is essentially im-portant to understand the role of one protein in certain dis-ease. Role of MyH8 in different pathways has been assessed using Reactome Event Hierarchy [25].

Stability Analysis

Amino acid substitution may lead to protein destabiliza-tion or some abnormal biological functions. We used ma-chine learning algorithm MuStab to predict the stability of the protein upon amino acid substitution [26]. We also de-termined change in Gibbs free energy of MyH8 protein upon mutation (i.e., G, the change in protein stability), using i-mutant 2.0 server, which is based on support vector machine, that predicts the protein stability changes on single point mutation [27].

Homology Modeling and Refinement

Since, the region up to 844 residues of MyH8 contains both nucleotide-binding site and the conserved residue

Arg674, therefore we generated a model up to 844 residues. Secondary structure of MyH8 was predicted using the Psipred program [28]. We considered the information ob-tained from the secondary structure of protein to improve the sequence alignment between the target and template protein sequences. MODELLER 9.11 was used to build the model of MyH8. To prepare the input file for MODELLER, FASTA sequence of MyH8 was converted to PIR format. The atomic coordinates of the template structure from the organism Gal-lus gallus were retrieved from the protein data bank [29] using the template structure of myosin sub-fragment at 2.8Å resolution (PDB ID: 2MYS) [30, 31]. The model of the tar-get sequence and mutated sequence were generated by MODELLER, that satisfies the spatial restraint to build the model. We computed the energies of the model and the mu-tant in vacuo with the GROMOS 96 using Swiss-PdbViewer 4.1.0 [32]. The energies thus obtained were high, so the structures were subjected to energy minimization using Swiss-PdbViewer 4.1.0. Comparative analysis of the DOPE score of template structure, model structure and refined model structure was carried out using the gnuplot version 6.4.6. Finally, the models were checked using Profile-3D [33]. The Profile-3D method estimates the compatibility of an amino acid sequence with a known three dimensional

Table 1. Intramolecular interactions offered by the Arg674.

Source Atom (ARG674) Target Atom Distance (Å)

N 675(CYS)N 3.43

N 124(CYS)O 3.28

N 673(VAL)CB 3.33

N 673(VAL)CG1 3.17

N 673(VAL)O 2.78

N 673(VAL)N 3.47

CA 673(VAL)O 2.78

CB 675(CYS)N 3.38

CD 699(ASN)O 3.45

NE 673(VAL)O 3.04

NH1 699(ASN)O 3.37

NH1 123(PHE)CE1 3.05

NH2 177(LEU)O 3.02

NH2 673(VAL)O 3.39

C 673(VAL)C 3.42

C 675(CYS)C 3.27

O 126(THR)N 2.91

O 675(CYS)CA 2.78

O 124(CYS)O 3.30

O 125(VAL)CA 3.29

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protein structure. This is especially useful in the final phase of the protein structure modeling.

Contact map of Arg674, an important residue for proper functioning of MyH8, is generated using contact program from the CCP4 suite [34, 35]. Intramolecular interactions of Arg674 in the modeled structure with its neighboring resi-dues within the range of 2.5 to 3.5 Å are described in Table 1. We also evaluated intramolecular interactions of Gln674 with its neighboring residues in the mutated model, which are presented in Table 2. We validated the model and the mutant structure for the stereochemical quality of the side chains using PROCHECK [36], to proceed further for dock-ing analysis.

Docking

In order to dock, the structure of ATP was obtained from HIC-Up database (http://xray.bmc.uu.se/hicup/ATP/ind-

ex.html). The nucleotide binding pocket of the modeled pro-tein structure was considered as a receptor. We used Auto-Dock 4.0 [37] for targeted docking of the modeled structures with ATP. Before the docking process, we first prepared the PDBQT files of the protein and the ligand, where we added total gasteiger charge of 0.50 to the ligand after detecting its root. We obtained eight rotatable bonds out of 32 bonds in ATP. To prepare a PDBQT file of the model structure gasteiger charge of 9.012 was added. As the ligand binds to the nucleotide binding region of the structure, we drew the grid around it. The grid box size was adjusted to 37.8782, 18.539, 111.136 Å in x, y and z axis, respectively. We chose genetic algorithm search parameter to search the optimal conformers in the grid box. Then we began docking and run the command on Cygwin, as a result we considered .dlg file of the ligand for further evaluation of contacts of ATP with the model structure. We also carried out docking of the mu-tated modeled structure with ATP by preparing the PDBQT file of mutated model and adding gasteiger charge of

Table 2. Intramolecular interactions offered by the Gln674.

Source Atom Target Atom Distance (Å)

N 675(CYS)N 3.42

N 125(CYS)O 3.27

N 673(VAL)CB 3.37

N 673(VAL)CG1 3.28

N 673(VAL)N 3.45

CA 673(VAL)O 2.76

CB 675(CYS)N 3.40

CD 673(VAL)C 3.23

CD 124(PHE)CD1 2.95

OE1 124(PHE)CB 2.84

OE1 673(VAL)C 3.12

OE1 124(PHE)CG 3.13

OE1 124(PHE)CD1 2.80

OE1 124(PHE)CA 3.49

NE2 124(PHE)CD1 2.93

NE2 124(PHE)CE1 3.31

NE2 672(PHE)CB 3.48

C 675(CYS)C 3.29

C 673(VAL)C 3.43

O 127(THR)N 2.96

O 675(CYS)CA 2.78

O 125(CYS)O 3.27

O 126(VAL)CA 3.34

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11.0113 followed by making of grid box of size 37.8782, 18.539, 111.136 Å in x, y and z axis, respectively and run-ning the commands on the Cygwin. The two docked files were compared for their intermolecular interactions. The docked complex was analyzed using PyMOL, CCP4 suite [34, 38] and Discovery Studio 3.5 [39].

RESULTS AND DISCUSSION

Sequence Analysis

The sequence of MyH8 (UniprotKB Id: P13535) was re-trieved and subjected to find the homologous sequences with known structure in the protein data bank (PDB-BLAST). The search gave 13 sequences with known crystal structure. A significant alignment was observed with mysoin protein (PDB ID: 2MYS) with E-values corresponding to zero (Fig. S1). We found 27 orthologous sequences of MyH8 and per-formed multiple sequence alignment using Discovery studio package and ClustalW, resulting in a significant sequence similarity among all sequences (Fig. 1). The segment of the sequence containing the Arg674 is highly conserved. The region from Tyr458 to Gly517 is highly conserved among all the aligned sequences except few sequences that substitute

Thr to Glu, Asn or Ala. Another highly conserved region that forms a helix starts from Leu658 and ends at Asn679. The region that forms a loop is part of the nucleotide binding pocket and is highly conserved that extends from His689 to Arg709.

Motif and Domain Search

Identification of motifs using Pfam protein family data-base revealed presence of five motifs in the MyH8 sequence. These motifs are myosin_head (91 to 769) which forms the motor domain, myosin_N (37 to 78) is a myosin N-terminal SH3-like domain, IQ (787 to 805) is a calmodulin-binding motif, AAA_16 (164 to 196) is an ATPase domain, AAA_22 (173 to 198) is an AAA domain, T2SE (163 to 200) is Type II/IV secretion system protein, AAA_29 (170 to 188) is a P-loop containing region of AAA domain [40]. We used SMART tool to identify the domain architecture of MyH8. We found three distinct domains in the MyH8, including myosin_N domain (37 to 78) which has an SH3-like fold. Many myosins contain this domain, although its function is still not known. Second domain is MYSc domain (82 to 782) which is the largest domain in MyH8 and is ATPases do-main. This portion is the molecular motor which is essential

Fig. (1). Multiple sequence alignment of MyH8 (residues 644-694) with orthologous sequences showing Arg674 is highly conserved (high-lighted in blue). The residue number are written at the top of the sequence. Secondary structure elements are shown at the top of amino acid sequences where loop residues are shown in black line, however the -helix is shown in red rectangle and -strand represented as blue col-ored arrow. The alignment file was obtained using ClustalW program.

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for the muscle contraction that consists of cyclic interaction between thick and thin filaments of the myofibril. The third domain is an IQ domain (783 to 805) which is short calmodulin-binding motif consisting of conserved Ile and Gln residues. The domain architecture is described in (Fig. S2).

Functional Networking Partners of MyH8

Proper functioning of MyH8 requires interaction with numerous proteins. Identification of interaction of MyH8 with other proteins have been predicted by STRING [41]. The interacting network of functional proteins with MyH8 has been represented in (Fig. 2). We categorized the proteins that play important functional partners of MyH8 into four main groups. These groups are: myosin light chain, myosin heavy chain, cardiac muscle proteins and actins. Functional proteins in the group of myosin light chain is MYL4 (My-osin, light chain 4), a regulatory light chain of myosin. While MYL1(Myosin, light chain 1) is an alkali protein present in the skeletal muscles. MYL4 and MYL1 do not bind to cal-cium. The third protein in this group is MYLPF (myosin light chain, phosphorylable, fast) is also present in the skele-tal muscle. Proteins that belong to the second group of my-osin heavy chain are the skeletal muscle proteins such as

MYH1 (Myosin, heavy chain 1), MYH2 (Myosin, heavy chain 2) and MYH13 (Myosin, heavy chain13). These pro-teins are present in adults and are essential for muscle con-traction. MYH2 (Myosin, heavy chain 2) is also required for cytoskeleton organization. We identified two proteins in the third group of cardiac muscle protein. These are MYH7 (Myosin, heavy chain 7), a beta cardiac muscle protein and MYH6 (Myosin, heavy chain 6) which is an alpha cardiac muscle protein. Both are essential for muscle contraction [42]. Actins, the fourth group of functioning partners of MyH8 include two proteins namely ACTB (actin beta) and ACTG1(actin gamma1). Actins are the highly conserved proteins that are involved in various types of cell motility [43].

Pathways Analysis

Usually protein does not work alone in any cellular activ-ity. Hence, they need some partner proteins to participate in various biological pathways. It is important to understand a disease from a perspective of its involvement in certain pathways. We identified pathways in which MyH8 is essen-tially involved. The pathway "Muscle contraction" retrieved from the Reactome Event Hierarchy [44, 45], explains the processes by which calcium binding triggers actin-myosin

Fig. (2). Showing interactions of MyH8 with other functional proteins, predicted by STRING.

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interactions and force generation in smooth and striated muscle tissues. The actin-myosin complex activity carries out the conversion of chemical energy into mechanical en-ergy, thereby generating force through hydrolysis of ATP.

MyH8 was also found to be involved in the inhibitory ac-tion of lipoxins on neutrophil migration (http://pathway-maps.com/maps/). Lipoxins are bioactive signaling mole-cules (eicosanoids) that robust anti-inflammatory actions, including weakening of neutrophil adhesion to endothelial cells. The myosin II function is governed by phosphorylation of the myosin regulatory light chain by myosin light chain kinases (MLCK) that increases myosin ATPase activity and polymerization of actin cables. This results in the generation of contractile force necessary for cell motility as described in (Fig. S3).

Stability Analysis

Analysis by Mustab shows decrease in protein stability upon single mutation in the sequence at pH 7.0 and 25°C.

G change in protein stability analyzed by i-mutant2.0 at pH 7.0 and 25°C is -1.78 kcal/mol.

Structure Prediction

Secondary structure prediction of MyH8 suggests the presence of 34.47% of -helices, 10.78% of -strands and 45.73% of loops (Fig. S4). The identified helices and strands are corresponding to the topology of myosin. Due to the sig-nificance of mutation in MyH8 gene causing TPS, it is es-sentially important to do the structure analysis of both wild type and Arg674Gln mutant of MyH8. The refined model was validated through PROCHECK to check the stereochemical quality of the protein chains in model. Using PROCHECK, we found that 90.6% of the residues are pre-sent in the favored region, and only 0.5% of the residues are found to be present in the disallowed region of the Ramachandran Plot (Fig. S5). The energies of models were high and hence we minimized the energy of models (Table 3). Similarly the model build for the mutated sequence was analyzed. Model with a lowest DOPE Score of -90241.01563 was selected for validation.

The region from the residues 624 to 648 was observed to possess high DOPE Score values. This region forms the dis-ordered loop in the model, coordinates of this region are missing from the x-ray structure of the template. We tried to improve this region by the process of loop refinement and then generated the graph of the DOPE score, as shown in (Fig. S6). We generated five models for the mutant and se-lected the best model based on molpdf and DOPE score. The best mutant model was observed to have the low dope score throughout the whole structure. A graph of the Dope score

and mutant sequence was generated using gnuplot, as pre-sented in (Fig. S7). The model showing a close similarity with the template structure with RMSD of 0.716 Å2. A su-perimposed structure of the model and the template are pre-sented in (Fig. 2), clearly indicating the structural similarity.

Structure Analysis

We successfully modeled 844 out of 1937 residues of MyH8. The refined structure clearly resembles with myosin (Fig. 3). The overall structure is composed of a long helix which extends from the thick part of the head down to the carboxy terminus of the heavy chain, constituting the light chain binding region. The entire thick portion of the MyH8 head constitutes heavy chain that contains both the nucleo-tide-binding and actin-binding regions. These binding sites are located on opposite sides of the protein. The topology of the -sheet motif of the modeled structure closely resembles to the myosin structure with strands one and six of the -sheet run in the opposite direction to the other five strands. While, the modeled structure has one more -sheet, the sev-enth -sheet, that runs parallel to the other five strands. The central strand corresponds to the strand-loop-helix binding motif, which has the sequence GESGAGKT, a p-loop motif found in many nucleotide binding proteins [30]. It was re-ported that the lysine residue in the walker A motif is crucial for the nucleotide-binding [46]. On crossing the width of the modeled structure a small four-stranded anti-parallel sheet motif (Lys36 to Met81) is protruded from the rest of the head and is observed to be independent. This motif corre-sponds to the six-stranded motif of myosin. Although its function is not determined but seems to be an unessential domain for motility because it is missing in several single-headed myosin I-type molecules [47].

This motif of MyH8 is followed by the three strands of the large -sheet motif that are connected by a series of -helices. The first two strands extend from Tyr117 to Tyr119 and from Cys124 to Val127 and are connected by a -turn. There are three short helices prior to the fourth -strand in the sheet that extends from Gln174 to Gly180. The third strand belongs to the carboxy-terminal of the heavy chain fragment. The fourth strand which is the central strand, pre-cedes the phosphate binding loop and is followed by a helix, Lys186 to Ile200 forming the base of the nucleotide binding pocket. At the far end of the active site pocket a loop con-taining seven charged residues are observed between Glu204 and Gly217. This loop is considered a flexible loop. After this loop, a helix (Leu219 to Gly234) forms part of the nu-cleotide-binding pocket (Fig. 4).

Intra-molecular interactions of Arg674 of the modeled structure were analyzed using contact program of the CCP4

Table 3. Energies of the model and mutant structure computed using Swiss-PdbViewer 4.1.0.

Energy of the Model Energy of the Mutant

Before minimization 71018.852 590.020

After minimization -23185.736 -26285.818

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Fig. (3). Cartoon representation of MYH8 modeled structure overlaid with the template structure (PDB ID:2MYS). Three distinct domains (myosin_N domain: Asn37 to Val78), (MYSc domain: Asn82 to Asp782) and (IQ domain: Lys783 to Arg805) are shown in light orange, light pink and light blue, respectively. The long -helix extending from the head portion of the model corresponds to the binding site for the light chains. The diagram was drawn in PyMOL, a visualization software.

Fig. (4). Cartoon diagram represents MYSc domain containing Arg674 (stick, light blue). -pleated sheets are shown as palecyan arrows, alpha helices in limon color. Showing P-loop of the -pleated sheet adjacent to the Arg674.

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A

B

Fig. (5). Representation of interactions of the conserved residue Arg674 and its mutant Gln674 in the modeled structures. Arg and Gln resi-dues are shown in ball and stick. (A) Residues interacting with Arg674 are shown in stick (color: C=purple; N=blue; O=red and S=orange), hydrogen bonded residues are shown in color (C=green; N=blue; O=red and S=orange) in black line. (B) Residues interacting with Gln674 are shown in stick (color: C=purple; N=blue; O=red and S=orange).

suite. 20 atoms of eight residues have been identified within 2.5 to 3.5 Å distance with Arg674 involved in forming intra-molecular interactions. When Arg674 is mutated to Gln674,

23 atoms of eight residues forms close intra-molecular inter-actions with Gln674 within 2.5 to 3.5 Å (Fig. 5). Our obser-vation reveals presence of two hydrogen bonds at Arg674 in

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the wild type model. Mutated model has no hydrogen bond with any other residue at Gln674 position. A change in the hydrogen bonded interactions reveals significant change in the stability of MyH8 protein after the mutation from Arg to Gln at 674 position.

Docking of ATP

To predict the bound conformation and the binding affin-ity of ATP to the MyH8, we carried out docking studies. The receptor, MyH8 is held rigid during the docking process and

ligand atoms were movable. Finally, the docked complex of MyH8 with ATP was selected by the criteria of interacting energy combined with the geometrical matching quality. It is well stated that hydrogen bonds play an important role for structure and function of proteins [48]. Hence, we analyzed number of hydrogen bonds between ATP and MyH8. The docked structure shows that the complex is stabilized by five hydrogen bonds between ATP and Arg192 present at the base of the nucleotide binding pocket. Two important hydro-gen bonds are formed with Arg674, Arg674-Val673 and Arg674-Leu177 in the MyH8 (Fig. 5). Binding energy for

A

B

Fig. (6). Representation of ATP docked in the nucleotide-binding pocket of MYH8 structure. Ball and stick representation of ATP colored according to the atom type (C=Green; N=Blue; O=Red and S-Orange). Amino acid residues forming hydrogen bonds with ATP are colored according to atom type (C=Cyan; N=Blue and O=Red). Other interacting residues with the ATP are colored according to atom type (C=Magenta; N=Blue and O=Red). Hydrogen bonds are shown in black lines. (A) ATP docked at the nucleotide binding pocket of modeled structure, forming hydrogen bonds with Arg192. (B) Docked structure of mutated sequence with ATP, forming hydrogen bonds with Arg193 and Lys192.

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ATP with model in the docked structure is -1.64 kcal/mol. Similarly, we performed docking for the Arg674Gln mutant structure with the ATP, and extensively analyzed. Binding energy for ATP with mutant in the docked structure was ob-served to be -0.54 kcal/mol. It is interesting to note that the mutated residue, Gln674 is not forming hydrogen bond with any other residue in the structure. However, ATP forms five hydrogen bonds with Lys192 and Arg193 in the case of Arg674Gln mutant (Fig. 6). Aromatic amino acids are capa-ble of forming cation- interactions that are strong, non-covalent interactions contributing to the stability of protein secondary structure and drug-receptor interactions [49]. Cation- interactions arises between aromatic amino acids and Lys/Arg. Phe 492 is forming cation- interaction with Arg674. The mutated residue Gln674 is not capable of form-ing cation- interactions. All these findings suggest the sig-nificant role of Arg674 in the stability of MyH8 protein and its subsequent role of in the muscle contraction.

CONCLUSION

We performed sequence analysis of MyH8 to establish its role in TPS. We identified five motifs and three distinct do-mains in MyH8. The structure analysis of wild type MyH8 and its Arg674Gln mutant (known to be responsible for TPS), discovered several novel characteristics. Absence of cation- interaction in mutant docked model suggests a de-crease in the stability of protein secondary structure. Muta-tion of Arg674Gln displays absence of hydrogen bonds, thus decreasing the stability of the protein. Mutation to Gln674 has led to a decrease in protein stability. Docking studies for the two structures highlights interaction of ATP with resi-dues forming hydrogen bond and the stability of the complex and the significance of the nucleotide binding pocket. Our structure based prediction provides many new insights to our understanding of the role of MyH8 in TPS.

ACKNOWLEDGEMENTS

We sincerely thank University Grants Commission for financial assistance (Project no: 40-201/2011 (SR)).

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

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Received: May 16, 2014 Revised: July 04, 2014 Accepted: July 15, 2014