Journal of Theoretical Biology 374 (2015) 107–114

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Journal of Theoretical Biology journal homepage: www.elsevier.com/locate/yjtbi

Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations Faez Iqbal Khan a,b, Algasan Govender b, Kugen Permaul b, Suren Singh b, Krishna Bisetty a,n a b

Department of Chemistry, Durban, Steve Biko Campus, Durban University of Technology, Durban, South Africa Department of Biotechnology and Food Technology, Steve Biko Campus, Durban University of Technology, Durban, South Africa

H I G H L I G H T S

 Chitinase gene cloned and expressed from Thermomyces lanuginosus SSBP.  3D structure predicted and analyzed by docking and molecular dynamics simulations.  Chitinase was found to be stable and functionally active at higher temperatures.

art ic l e i nf o

a b s t r a c t

Article history: Received 20 January 2015 Received in revised form 2 March 2015 Accepted 27 March 2015 Available online 8 April 2015

Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K. & 2015 Published by Elsevier Ltd.

Keywords: Chitin TIM-barrel MD simulations Stability Molecular docking

1. Introduction Chitin is a natural biopolymer composed of repeating units of N-acetyl-β-D-glucosamine and primarily forms the structural component of protective biological matrices such as fungal cell walls and exoskeletons of insects and arthropods (Aam et al., 2010). Chitindegrading enzymes (chitinases) play a significant role in the defense against chitin-containing parasites by hydrolyzing the β-1,4-linkages in chitin (Bucolo et al., 2011; Lobo et al., 2013). Chitinases have been a focus of research in the past few years due to their vast array of biotechnological applications, especially in the field of agriculture for Abbreviations: LB, Luria Bertani; SSBP, Suren Singh Bernard Prior; cDNA, complementary deoxyribonucleic acid; RMSD, root mean square deviation; SignalP, signal peptide; MD, molecular dynamics; GROMACS, GROningen MAchine for Chemical Simulations; OPLS-AA/L, optimized potential for liquid simulations/all atoms; DS, Discovery Studio n Correspondence to: Department of Chemistry, Durban University of Technology SB Campus, POBOX 1334 Durban 4000. E-mail address: [email protected] (K. Bisetty). http://dx.doi.org/10.1016/j.jtbi.2015.03.035 0022-5193/& 2015 Published by Elsevier Ltd.

bio-control of fungal phytopathogens (Hamid et al., 2013). Since the effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance, chitinases offer a potential alternative to the use of chemical fungicides as well as anti-biofouling agents (Herrera-Estrella and Chet, 1999). The thermostable enzymes isolated from thermophilic microorganisms have gained widespread attention in industrial, medical, environmental and biotechnological applications due to their inherent stability at high temperatures and wide range of pH optima (Maheshwari et al., 2000; Meng Zhang et al., 2014; Stephens et al., 2014). However, despite their huge potential, the precise three-dimensional structure of most of the chitinases, including those isolated from Thermomyces lanuginosus (Duo-Chuan, 2006) is not fully characterized. Hence, the main focus of the present study was to gain a better understanding of the structural features of chitinases obtained from this thermostable fungus using both experimental and computational techniques, and their relationship with their activity profiles. In this work, a novel chitinase II gene from T. lanuginosus was expressed in E. coli. The structure of chitinase II was predicted using homology modeling and molecular dynamics (MD)

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simulations methods. The generated (3D) structures were refined and the best reliable models were selected and subjected to MD simulations to better understand the details of protein conformation and stability as a function of time.

2. Materials and methods 2.1. Strains, plasmids and growth conditions T. lanuginosus SSBP (Singh et al., 2000) was grown at 50 1C on potato dextrose medium (Merck), sub-cultured every two weeks and stored at 4 1C. E. coli BL21 (Stratagene) was grown and maintained on Luria Bertani (LB) medium (10 g/l peptone, 5 g/l yeast extract powder, 10 g/l NaCl and 15 g/l technical agar). Transformed E. coli was maintained on LB medium supplemented with 100 μg/ml ampicillin and grown at 37 1C. For short term storage, sub-culturing was done every two weeks, whilst for long term storage cultures were supplemented with 15% glycerol and stored at  70 1C. The pET21c cloning vector (Novagen) was used in this study. 2.2. Chitinolytic plate enzyme assays Chitinase activity was assayed with glycol chitin as a substrate. Glycol chitin was prepared according to the methods of Trudel and Asselin (1989) and Lee et al. (2007). Glycol chitin at different concentrations was added to melted 1% agarose and poured into petri plates. Wells were punched into the agarose medium after solidification. 400 ml of supernatant was loaded into different wells in the plate with the negative control being chitinase production medium, which was then incubated for 2 h at 50 1C. The plate was then stained with 2% Calcofluor White M2R stain (Fluka) for 1.5 h and washed with distilled water for 2 h, then observed under UV light to determine whether clear zones can be observed around the wells.

incubation of ligation mixture at 4 1C. The ligated product was transformed into competent E. coli BL21 and selected on LB agar plates containing ampicillin using standard protocols. Positive clones were confirmed by colony PCR. 2.5. Sequence analysis The 343 residue long amino acid sequence of chitinase II from T. lanuginosus SSBP was analyzed using several available bioinformatics tools such as BLAST (Altschul et al., 1990), HMMER (Finn et al., 2011) and FASTA server (Pearson and Lipman, 1988). BLAST searches the non-redundant protein sequences (nr) database and hits with an e-value (o0.0005) and sequence identity (420%) were considered as matches. The domain analysis was performed using InterProScan 5 (Jones et al., 2014), SMART (Schultz et al., 2000), SYSTERS (Schultz et al., 2000) and ProtoNet (Rappoport et al., 2012) in order to identify the function more precisely. InterProScan 5 searches the similar signature in the interpro consortium databases. PTMcode (Minguez et al., 2013) was used to detect presence of any possible posttranslational modification sites in the protein sequence. SMART was further used to annotate the genetically mobile domains in the sequence of chitinase II and SYSTERS use the clustering based algorithms for the function prediction of proteins. The Classify Your Protein module of ProtoNet was used to analyze the protein family of chitinase II. The sequence based secondary structure was analyzed using Psipred (McGuffin et al., 2000). The presence of a signal peptide in the sequence framework of chitinase II was predicted using artificial neural network algorithm of SignalP 4.0 (Petersen et al., 2011) and Signal-CF (Chou and Shen, 2007; Shen and Chou, 2007). Signal-CF performed the best among the existing signal peptide predictors, particularly for long signal peptides (Hiss and Schneider, 2009). Protein glycosylation is significant for secretion, localization and stability of protein. Thus, glycosylation sites were predicted using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). 2.6. Structure prediction and evaluation

2.3. Total RNA isolation from Thermomyces lanuginosus SSBP and first-strand cDNA synthesis 1 ml of an aqueous spore suspension (1  106 spores/ml) was inoculated into chitinase production medium [10 g/l colloidal chitin; K2HPO4, 0.87 g/l; KH2PO4, 0.68 g/l; KCl, 0.2 g/l; NH4NO3, 1 g/l; MgSO4, 0.2 g/l; yeast extract, 4 g/l; pH 6.5] (Guo et al., 2005) and incubated at 50 1C on a rotary shaker at 150 rpm. Mycelia were harvested by centrifugation following 12, 24 and 48 h incubation and stored at 70 1C. Total RNA was isolated using the TRIzol reagent and the integrity was confirmed by electrophoresis (0.8% agarose gel) and NanoDrop 1000 spectrophotometer (OD26041.8) analysis. The firststrand cDNA was synthesized from 5 mg total RNA using the iScriptTM cDNA synthesis kit (Bio-Rad). 2.4. PCR amplification and cloning Primers were designed using Oligo version 6.0 software (Rychlik, 2007), based on chitinase genes identified in the currently available annotated genome sequence data of T. lanuginosus SSBP (GenBank Accession no. KJ740647) and included the NheI and XhoI restriction sites. PCR amplification was done under the following conditions: initial denaturation at 98 1C for 30 s, cyclic denaturation at 98 1C for 10 s, primer annealing at 55 1C for 20 s, extension at 72 1C for 30 s (30 cycles) and final extension at 70 1C for 7 min. The PCR product was confirmed by 1% agarose gel electrophoresis and purified using the DNA Clean and Concentrator Kit (Zymo Research). Purified DNA was digested with restriction enzymes NheI and XhoI (Fermentas) and once again purified using the DNA Clean and Concentrator Kit (Zymo Research). This was then ligated into the pET21c vector by overnight

The knowledge of protein three-dimensional (3D) structures is vitally important for rational drug design (OuYang et al., 2013). Although X-ray crystallography is a powerful tool in determining protein 3D structures, it is time-consuming and expensive, and not all proteins can be successfully crystallized (Berardi et al., 2011). Membrane proteins are difficult to crystallize and most of them will not dissolve in normal solvents. Therefore, so far very few membrane protein structures have been determined (Call et al., 2006, 2010). NMR is indeed a very powerful tool in determining the 3D structures of membrane proteins, but it is also time-consuming and costly. To acquire the structural information in a timely manner, a series of 3D protein structures were developed by means of the homology technique (Chou, 2004, 2005; Wang et al., 2007) and from a comprehensive review (Chou, 2004), it was found very useful for drug development. In view of this, the homology technique was also adopted to develop the relevant protein 3D structures for the current study. The 3D structure of chitinase II was predicted using homology and ab initio methods. The structural homolog search was performed in the Protein Data Bank (PDB) (Berman et al., 2000) using PSI-BLAST module in Discovery Studio (DS) 4.0 (Accelrys Software Inc. 2013), HHpred (Soding et al., 2005), HMMER (Finn et al., 2011) and Phyre (Kelley and Sternberg, 2009). A BLAST (Altschul et al., 1990) search with identity 430% was considered as the suitable template for the structure prediction of the chitinase II. The HHpred identified structural homologs in the PDB, structural classification of proteins (SCOP) and CATH. Furthermore, HMMER were used to detect reliable homologs in the databases by exploring the modules such as hmmsearch, hmmscan, jackhammer and phemmer. Similarly, Phyre a server based on profile–profile matching algorithm was used to enhance the

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accuracy of the similarity search. The fold recognition methods were used to optimize the sequence–structure alignment, which was further utilized to develop the three dimensional models using MODELLER (Eswar et al., 2008) module in DS 4.0. The homology modeling is based on the alignments of the studied proteins; therefore, the unaligned regions were modeled using the ITASSER server (Roy et al., 2010). The most reliable models were evaluated on the basis of root mean square deviation (RMSD), TM score and DOPE Profile. The selected models were further refined using SCWRL 4.0 (Wang et al., 2008) and CHARMm (Vanommeslaeghe et al., 2010) energy minimization using ChiRotor algorithm of DS. The GROMOS (van Gunsteren et al., 1996) algorithm implemented in DeepView (Kaplan and Littlejohn, 2001) was used for energy minimization of the predicted chitinase II structure. The 3D models were evaluated using the modules of the SAVES server (http://nihserver.mbi.ucla.edu/ SAVES/) such as PROCHECK, WHAT_CHECK, VERIFY3D, ERRAT and PROVE modules. 2.7. Molecular dynamics Many remarkable biological functions in proteins and DNA and their profound dynamic mechanisms, such as switch between active and inactive states (Wang and Chou, 2009), cooperative effects (Chou, 1989), allosteric transition (Chou, 1987; Wang et al., 2009), intercalation of drugs into DNA (Chou and Mao, 1988), and assembly of microtubules (Chou et al., 1994), can be revealed by studying their internal motions as elaborated in a comprehensive review (Chou, 1988) and summarized in a recent paper with the title of ‘Theoretical and Experimental Biology in One’ (Sheng-Xiang Lin, 2013). Likewise, to really understand the action mechanism of receptor–ligand binding, we should consider not only the static structures concerned but also the dynamical information obtained by simulating their internal motions or dynamic process. To realize this, the MD simulation is one of the feasible tools. MD simulation methods were performed on chitinase II at 300 K, 325 K, 350 K and 375 K in order to investigate stability profile (Anwer et al., 2013). The MD simulations were performed at the molecular mechanics level implemented in the GROMACS 4.6.5 computer program (Van Der Spoel et al., 2005) using the all atom functions by OPLS (optimized potential for liquid simulation). The proteins were soaked in a cubic box of water molecules with a dimension of 10 Å i.e. setting the box edge 10 Å from the molecule periphery using the editconf module for creating boundary conditions and genbox for solvation. The spc216 template was used to solvate the proteins. The charges on the protein were neutralized by the addition of Na þ and Cl  ions to maintain neutrality. The system was then minimized using 1500 steps of steepest descent. The temperature of all the systems was subsequently raised from 0 to 300 K, 325 K, 350 K and 375 K during their equilibration period (100 ps) at a constant volume under periodic boundary conditions. Equilibration was performed in two phases: NVT ensemble (constant number of particles, volume, and temperature at 100 ps) and NPT ensemble (constant number of particles, pressure, and temperature at 100 ps). After the equilibration phase, the particlemesh Ewald method (Norberto de Souza and Ornstein, 1999) was applied and the production phases consisting of 10 ns were performed at 300 K, 325 K, 350 K and 375 K. The resulting trajectories were analyzed using g_rms, g_rmsf, g_energy and do_dssp utilities of GROMACS. All the graphic presentations of the 3D model were prepared using Discovery Studio 4.0 and VMD (Visual Molecular Dynamics) (Humphrey et al., 1996).

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metaPocket 2.0 (Zhang et al., 2011), COFACTOR (Roy et al., 2012) and COACH (Yang et al., 2013). The metaPocket 2.0 algorithm uses LIGSITEcs, PASS, Q-SiteFinder, SURFNET, Fpocket, GHECOM, ConCavity and POCASA predictors to identify pocket sites. The COFACTOR identifies the template proteins of similar folds and functional sites by threading the target structure through three representative template libraries with known protein–ligand binding interactions, Enzyme Commission number or Gene Ontology terms. The COACH module was based on the binding-specific substructure comparison (TM-SITE) and the sequence profile alignment (S-SITE), for complementary binding site predictions. The docking studies were further done with allosamidin as ligand, a reported chitinase inhibitor (Suzuki et al., 2006) to visualize the interaction with the predicted active pocket site. Molecular docking can provide useful information for drug design as demonstrated by a series of previous publications (Cai et al., 2011; Du et al., 2005, 2009, 2010). The receptor and the ligands were optimized using various protocols available in DS such as ligand receptor preparation, energy minimization and binding site characterization. Docking studies were performed using CDOCKER (Wu et al., 2003), a CHARMm-based method that generated highly accurate docked poses, of which the 10 best hits were selected and the docking energies were compared. The results were further validated via AutoDock 4.2 (Norgan et al., 2011).

3. Results and discussion 3.1. Amplification and cloning In a previous study, it has been shown that the T. lanuginosus SSBP does not possess cellulolytic activity (Singh et al., 2003), therefore expression of the native chitinase II enzyme in T. lanuginosus was confirmed by clear zone in the chitinolytic plate assay (Fig. 1). The chitinase II gene was successfully amplified by PCR using cDNA as template and purified from agarose (Fig. 2). The size of the product was 1032 bp. The gene was successfully cloned into the E. coli BL21 expression vector, pET21c which was confirmed by colony PCR and enzymatic digestion. The predicted size of chitinase II protein was 36.6 KDa (Fig. 3). There is only one potential N-linked glycosylation site at position 258 occupied by asparagine as predicted by NetNGlyc 1.0 server. 3.2. Sequence analysis The ProtParam revealed that chitinase II DNA sequence encodes a 343 amino acid protein of molecular weight 36.6 kDa with theoretical pI of 4.45 and estimated half-life of more than 20 h (yeast, in vivo). The BLAST search shows that the given protein is highly similar to fungal

2.8. Active site prediction and docking studies The active site was predicted by various bioinformatic tools as well as by comparing the chitinase II amino acids sequence with template and other similar proteins. The active site pockets were predicted by

Fig. 1. Chitinolytic enzyme plate assay well (1, 2, 3, 4 and 5) showing zones of hydrolysis. Well (0) negative control chitinase II production medium.

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class III chitinase with highest identity of 65% to Byssochlamys spectabilis chitinase III having an e value of 3e 157. The InterProScan identifies that the sequence contains the domain similar to glycoside

1

2

3

4

1032 bp

1000 bp

Fig. 2. Agarose gel showing chitinase II PCR product. Lane 1: 1 kb DNA marker, Lane 3, 4: PCR product.

1

2

3

4

5

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66 51 35

36 kDa

25 14 Fig. 3. SDS-PAGE analysis of chitinase II protein stained with Coomassie Brilliant Blue G 250 (sigma). Lane 1: protein marker, lane 5: E. coli cell pellet, lane 7: 36 KDa chitinase II protein.

hydrolase, family 18 with active site ranging from 168 to 176 amino acids. However the domain annotation has various limitations as demonstrated by a stringent domain–domain interaction based approach based on high sequence similarity between template domain instances and query domain instances (Zhou and Wong, 2011; Zhou et al., 2013). Glycoside hydrolase family 18 enzymes, hydrolyzes the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. No post-translational modification sites were observed using PTM code server. The SignalP and Signal-CF prediction revealed presence of a putative N-terminal signal peptide of 23 amino acids with a predicted cleavage site located between A23 and G24 in chitinase II. These results confirm secretory nature of this enzyme. The NetNGlyc 1.0 server analysis showed the presence of one potential N-linked glycosylation site at position 258 occupied by asparagine. The secondary structure topology predicted by the PSIPRED server identified chitinase II may consist of eight α-helices and seven β-strands connected via loops.

3.3. Structure prediction and evaluation The HHpred and PSI-Blast identifies Saccharomyces cerevisiae chitinase (PDB Id: 2UY2) as the suitable template for the structure prediction of chitinase II. The sequence and structure alignment shows 43% identity. The MODELLER aligned the template structure and query sequence and finally generated the 3-D models by satisfying the spatial restraints. The predicted structures of chitinase II that showed low violations of restraints are considered to be more precise. 20 models were generated and evaluated on the basis of RMSD, TMscore, DOPE profile and Molecular Probability Density Function (Mol PDF). The predicted model is highlighting the overhanging sequence which is further modeled using ab intio protocol of the I-TASSER (Roy et al., 2010). DOPE energies of the model and the template structures were compared, which revealed loop region from the residue 24 to 105 of the model have higher DOPE energy than the template. The generated model was optimized using the CHARMM 22 force field

Fig. 4. (A) Topology map of chitinase II indicating a conserved α/β TIM-barrel present in the framework. (B) Secondary structural elements of chitinase II obtained using PDBsum indicating 8 parallel β strands, 2 anti-parallel β strands, 10 helices and three possible disulfide cysteine bridges (58Cys-113Cys, 92Cys-103Cys and 209Cys-238Cys).

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present in the DS. The side chains of the model were refined using the SCWRL4 package. Then the energy minimization of the 3-D structure was performed in order to avoid bad molecular contacts by using the Deepview that contain steepest descent protocol implemented in GROMOS. The minimized energy for the optimized model was

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observed to be  5523.179 KJ/mol. The evaluations of the models were performed based on their RMSD, DOPE profile and TM-score using the comparison with the template. The RMSD between the two structures was found to be 0.945 and TMscore of 0.1719 indicating that both belong to the same fold. Further, DOPE energies of the model and the template structures were compared using gnuplot, which revealed

Fig. 5. Structure of chitinase II indicating an overall TIM-barrel (represented by Pymol).

Fig. 6. RMS deviation values at different temperatures as a function of time. Black, red, green and blue represent RMS deviations obtained at 300 K, 325 K, 350 K and 375 K, respectively. The duration of each simulation is 10 ns. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Average RMS fluctuation values as a function of amino acid sequence. Values were calculated with the use of Cα atoms. Black, red and green color represent RMS fluctuation obtained at 300, 325 and 350 K, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Graphical representation indicating the stability of structural elements of chitinase III at (A) 300 K, (B) 325 K and (C) 350 K respectively.

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a loop region from the 24 to 105 of the model have higher DOPE energy than the template. The models were further evaluated using SAVES. The Procheck shows 98.3% of the residues in allowed region of the Ramachandran plot. Verify_3D shows 88.66% of the total residues had an averaged 3D–1D score of 40.2 indicating the reliability of the model prediction. The overall quality factor score predicted by Errat was 63.582. The topology for the given chitinase II structure was generated using PDBsum (de Beer et al., 2014) to understand the detailed structural features. The structurally conserved (α/β)8 TIMbarrel was observed in the framework of chitinase II (Fig. 4A). All TIM barrels known to date are enzymes with catalytic functions. The α/β barrel structure is probably one of the most striking structures in proteins. The right-tilted and right-handed crossover α/β barrel structure is energetically most stable type of structure (Chou, 2004). The chitinase II contains 18.7% beta strands (56 aa), 27.3% alpha helix (82 aa) and 0.9% 3-10 helix (3 aa). The structure shows the presence of 2 beta sheets which comprises 8 parallel strands, 2 anti-parallel strands, 1 beta hairpin, 10 helices with 9 helix–helix interactions and 6 characteristic beta–alpha–beta motifs that may be responsible for the activity of enzyme. Moreover, there are two disulfide cysteine bridges between 58Cys-113Cys and 209Cys-238 Cys. There is an extra possible chance of disulfide bond between 92Cys-103Cys in the structure. Introduction of a disulfide bond in the protein structure may be responsible for the thermostability (Yu et al., 2012). Chitinase II contains well defined TIM-barrel with a large α/β domain inserted into several loops (Fig. 5). The structure contains a shallow substrate binding groove as indicated after structural comparison with template. Chitinase II shows the presence of one conserved solvent exposed amino acid Trp307.

3.4. MD simulations The MD simulations of chitinase II at three different temperatures showed an acceptable stability profile at a temperature of 300 K, 325 K and 350 K, respectively, while instability was observed at 375 K, in keeping with the experimental data. The relatively stable nature of the protein was also observed by computing the average total energies. The average total energy for the chitinase II was found to

gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE gi|702076139|gb|AIW06012.1| 2UY2_A|PDBID|CHAIN|SEQUENCE

be  1,002,760 kJ/mol,  951,579 kJ/mol and  1,003,070 kJ/mol showing relatively stable nature of protein at 300 K, 325 K and 350 K respectively. Similarly, the RMSDs ranged from 0.5 nm, 0.4 nm and 0.3 nm, corresponding to 300 K, 32 K and 350 K, respectively (Fig. 6). However, the average RMSD of the chitinase II structure was found to be low at 350 K as compared to 325 K and 300 K. Initial fluctuations were observed at 300 K and thereafter a constant equilibrium was achieved. The slight fluctuations observed at 325 K and 350 K, clearly suggests the stable nature of the protein at higher temperatures. To evaluate the average fluctuation of each residue during the simulation, the RMSF of the Cα atoms of chitinase II from the initial structure were plotted as a function of residue number (Fig. 7). The RMSF of the Cα value of the chitinase II structure was comparable for each temperature. At 325 K, the loop forming residues ranging from 40 to 50 showed higher fluctuations. Similarly, the region spanning residues from 80 to 100 showed higher values of RMSF at 325 K and 350 K, while the coiled region of 240–250 residues displayed an elevated value at 350 K. However, the enhanced localized flexibility in the loop regions suggests a greater stability to the chitinase II enzymes at higher temperatures. The secondary structures observed during the MD simulation were highlighted by do_dssp module of GROMACS, suggesting that the global conformation of chitinase II is conserved with no significant changes in its secondary structure elements (Fig. 8).

3.5. Active site prediction and docking studies Using the information present in the literature and on the basis of the outputs obtained from the metaPocket 2.0, COFACTOR and COACH servers, the active pocket of the chitinase II may contain Tyr36, Phe70, Asp174, Glu176, Gln208, Gln232, Tyr234 and Trp307. The Glu176 was predicted to be essential for the activity of the chitinase II (Fig. 9). In order to validate the above predicted results the docking analysis was performed. The allosamidin and chitinase II were prepared for the docking using “Prepare ligands” and “prepare protein” protocols of DS. The preparation processes involve the adding of hydrogen in the structural framework of the respective molecules and cleaning of the geometry. Furthermore, for precise interaction at the predicted active

10 20 30 40 MPSFKSVVSSLPVILTALPSVQAGLDLSSTSNVVVYWGQNSAAASGGGPS -------------------------DRSANTNIAVYWGQNSAG------T * *:.:*:.********. : 60 70 80 90 QQPLATYCEDPNIDTLVMAFMTRINGAGGVPEINLANIGDSCGTFDGTNL QESLATYCESSDADIFLLSFLNQFPTLG----LNFANA--CSDTFS-DGL *:.******..: * ::::*:.:: * :*:** ...**. .* 110 120 130 140 KDCPQVGEDIKKCQSLGKTILLSIGGATYTEGGFQSAEAAEAGARMVWET LHCTQIAEDIETCQSLGKKVLLSLGGASGSY-LFSDDSQAETFAQTLWDT .*.*:.***:.******.:***:***: : *.. . **: *: :*:* 160 170 180 190 FGPVTNGDALRPFGDAVVDGFDLDFEATVSN-MVPFANTLRSLMDSDSSK FGEGT-GASERPFDSAVVDGFDFDIENNNEVGYSALATKLRTLF-AEGTK ** * * : ***..*******:*:* . . .:*..**:*: ::.:* 210 220 230 240 QYFLTAAPQCPFPDAANKEMLDGAVSFDAIWVQFYNNYCGVNSYPDNFNF QYYLSAAPQCPYPDASVGDLLENAD-IDFAFIQFYNNYCSVS---GQFNW **:*:******:***: ::*:.* :* ::*******.*. .:**: 250 260 270 280 NTWDDWAQNTSKNKNVKVLVGVPANTGAAGSGYLP-VDQLAPVIEHARTF DTWLTYAQTVSPNKNIKLFLGLPGSASAAGSGYISDTSLLESTIADIASS :** :**..* ***:*:::*:*..:.******:. .. * ..* . : 310 320 330 340 PSFGGVMMWDASQAYANDGFLSGIKSILGSVISRVKRMFFRRDFW 343 SSFGGIALWDASQAFSNELNGEPYVEILKNLLTSASQTA------ 294 .****: :******::*: . .** .::: ..:

50 19 100 62 150 111 199 159 249 205 298 255

Fig. 9. Structural analysis with template (PDB id: 2UY2) shows active site pocket in chitinase II contains 36, 70, 174, 176, 208, 232, 234 and 307 amino acid residues.

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pocket the “Current selection” module present in the “Define and Edit Binding Site” protocol was used. Then allosamidin is docked at the active site pocket of the chitinase II using CDOCKER protocol of the DS and AutoDock 4.2. The top 10 conformations of the docked allosamidin–chitinase II complex were selected for the evaluation. There are three stable conformations (pose1, 2 and 3) that confirmed the above predicted ligand binding site (Fig. 10). The docked pocket contains Ala127, Asp174, Glu176, Ala206 and Gln232. The CDOCKER energy for these predicted conformations was found to be  8.29987,  8.39712 and  10.6359 Kcal/mol, while CDOCKER interaction energy was calculated to be  29.1129,  29.3752 and  27.4068 Kcal/mol, respectively.

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While the docked complexes obtained from AutoDock 4.2 formed 7 hydrogen bonds with the residues Gln208, Cys209, Gln232, Tyr234, Asn235 and Asn236 with binding energy  5.14 Kcal/mol.

4. Conclusion The cloning and 3D structural analysis of chitinase II revealed several novel characteristics of the thermostable enzyme isolated from T. lanuginosus. The present work offers insights into the structural and dynamic features of a family 18 chitinase. The identification of the surface exposed residue Glu176 is anticipated to have a profound effect on the substrate binding. The detailed study of the 3D structure of chitinase obtained from T. lanuginosus has not yet been fully understood. In this regard, MD simulations provided valuable insights into the structure and stability of the protein. These simulations clearly suggest that the stability and characteristics of the secondary structures are maintained up to 350 K.

Acknowledgments The authors would like to express their acknowledgment to his colleagues Danish Idress from Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, India. We would like to express our gratitude to the Centre for high performance computing, an initiative support by the Department of Science and Technology of South Africa. References

Fig. 10. Possible docked positions of chitinase II with C Docker energy  8.29987,  8.39712,  10.6359 Kcal/mol and interaction energy of  29.1129,  29.3752,  27.4068 Kcal/mol, respectively, noting the shift in the key catalytic residues Ala127, Asp174, Glu176, Ala206 and Gln232 as determine by Cdocker of Discovery Studio.

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Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations.

Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at h...
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