insilico docking

nst Life Sciences and Bioinformatics ISSN: 0974-9179

http://www.nstjournal.com Open Access

Research Paper

In-Silico Modeling and Investigation of ATP Binding Pocket of An Algal Oil Producing Enzyme Raghunath Satpathy*, Rajesh ku.Guru, Rashmi R Behera and Aparajita P

Department of Biotechnology, MIRC LAB, MITS Engineering College, Rayagada, Odisha, India – 765 017.

(*Corresponding Author E-mail: [email protected]) (Received March 11, 2010; Revised and Accepted March 31, 2010)

ABSTRACT

Acetyl-CoA carboxyl transfersae (ACCase) is the key enzyme for the synthesis of oils in algae. The particular enzyme catalyses the reaction to form the Malonyl -CoA by binding with the substrate like ATP and Coenzyme A. The present work is an in-silico approach which deals with the Homology based modeling and identification of ATP binding residues of the enzyme ACCase of the biofuel producing algae Cyanidium caldarium. MODELLER program has been used for 3D modeling. The reliability of the model was verified on basis of PROCHEK, ERRAT, and, DOPE result. Then prediction of binding pocket and docking study show that the residues ILE 110, ARG113, LEU 296 are responsible for binding to ATP. The structural motif analysis for the protein sequence by EMBOSS show that Prosite motif present in the sequence is AGRR (110-113), which are in an agreement with previous findings. The stability of the docked structure is also checked by energy minimization by Chimera. Key words: Homology modeling; algae; docking; biofuel; binding site.

INTRODUCTION

The development of alternative energy sources such as biofuels are one of the most exciting and challenging area. So the effort has been put to produce biodiesel from the algal species [1].The red algae Cyanidium caldarium is one of the most preferable organism to produce the oil [2]. Considering the metabolic pathways of oil production in the algae, the Acetyl-CoA carboxyl transfersae (ACCase) enzyme plays a major role for oil production. It catalyzes the production of Malonyl Co- enzyme a using the substrate Acetyl -CoA and ATP [3].As the three dimensional structure of the enzyme is not present in PDB (Protein Data Bank), so an in-silico approach has been taken to predict the three dimensional structure by homology modeling method [4]. By this approach a valid structural model has been produced with the available template having 46% similarity. After model generation and validation an automated version of pocket finding search by CastP server [5] docking by Hex 5.0 version and structural motif analysis by EMBOSS tool [6] has been used to investigate the binding residues responsible for binding of ATP. Furthermore the in-silico structural analysis

shows that the residues like 110,113,296 are responsible for the binding to ATP. MATERIALS AND METHODS Sequence retrieval and 3D model building The sequence for the ACCase enzyme was retrieved from SWISSPROT database having ID O19903 [7]. Then with this query sequence a BLAST [8] search was performed against PDB (Protein Databank) to retrieve the corresponding template for the ACCase enzyme. The model was built by homology modeling and for this MODELLER 9v5 [9] program. The MODELLER program uses an automated approach to comparative protein structure modeling by satisfaction of spatial restraints (Fig.1). Briefly, the core modeling procedure begins with an alignment of the sequence to be modeled (target) with related known 3D structures (templates). This alignment is usually the input to the program. The output is a 3D model for the target sequence containing all main chain and side chain non-hydrogen atoms [10].

_______________________________________________________________________________________________________________________ Journal of Natural Science and Technology - Life Sciences and Bioinformatics, 2010, Vol. 2: Page No. 147-152 -147-

nst Life Sciences and Bioinformatics Raghunath Satpathy et al. http://www.nstjournal.com

Model validation The MODELLER generated structure was verified for the missing side chains by SCWRL4 tool [11] further verified by PROCHECK [12]. The PROCHECK programs provides the information about the stereo chemical quality of a given protein structure. The PROCHECK was used to generate Ramachandrans plot and the quality of the structure was computed in terms of % of residues in favorable regions, % of non Proline Glycine residues etc.The quality of structure was also further accessed by using ERRAT [13]. ERRAT is a protein structure verification algorithm that is especially well-suited for evaluating the progress of crystallographic model building and refinement. The program works by analyzing the statistics of non-bonded interactions between different atom types. A single output plot is produced that gives the value of the error function vs. position of a 9-residue sliding window. By comparison with statistics from highly refined structures, the error values have been calibrated to give confidence limits. This is extremely useful in making decisions about reliability. Then the backbone alignment and RMSD (root mean square deviation) study was performed by using PYMOL tool [14]. PyMOL is useful molecular modeling software allowing the visualization of three dimensional molecular structures as well as for the backbone alignment between the model and template. In-silico conserved motif analysis and binding pocket analysis of the model The refined validated model was then subjected to CastP server to observe the possible pockets in the protein.CastP server is a automated on line tool that predicts the possible pockets for along with the residue positions and surface area.Also to find out the conserved motif responsible for binding then protein sequence was searched in EMBOSS tool which scan a protein sequence with motif from Prosite database.

Docking study and energy minimization Docking was performed between the validated structure of ACCase enzyme and the energy minimized ligand by using HEX 5.0 tool [15]. HEX is an interactive molecular graphics program for protein-ligand docking, assuming the ligand is rigid, and it can superpose pairs of molecules using only knowledge of their 3D shapes. It is still the only docking and superposition program to use spherical polar Fourier (SPF) correlations to accelerate the calculations [16].The binding energy was computed and the ligand binding pattern was observed. Then a compared energy minimization was performed in the model alone and model with the docked ligand by Chimera tool for 100 steps to observe the stability in terms of potential energy.

RESULTS AND DISCUSSIONS Homology modeling of ACCase enzyme The sequence of 324 amino acid protein AcetylCo-enzyme carboxylase carboxyl transferase subunit alpha was obtained from SWISSPROT database. After the sequence searched in PDB (protein data bank) by BLAST the template PDB ID 2F9I [17] chain A was chosen as suitable one as it is having 46 % identity with the query sequence. Then MODELLER was used to generate the three dimensional structure (Fig.2) and the structural features were derived (Table 1). Overall six models were obtained the suitable model was chosen by lowest molpdf value. Refining and Validation of the Model After getting the model the missing side chains of the model was checked and managed by SCWRL4 software. The output PDB file from the software was then verified by backbone comparison between the template and refined model. The RMSD of backbone was calculated as 0.210 which is very much reliable. The modeler generated DOPE score was computed and analyzed. From the comparative chart (Fig.3) the peak is showing that there is no defect in the loop regions in the residues. So in the present case the loop refinement method is not considered for the model. To verify further the predicted structures, the coordinates of the predicted structure was fed into the ERRAT Protein Verification Server. The overall quality factor was obtained as 78.344 which are very much satisfactory (Fig.4). The stereo chemical quality of the structure was checked by PROCHECK tool. The Φ and Ψ distributions of the Ramachandran plots of non-Glycine, non-Proline residues are summarized (Fig 5). Altogether 92.3% of the residues were in favored regions (Table 2) so it can consider as a good model for further analysis. The overall G-factor used was computed as- 0.14. Docking study Docking of the ATP with the modeled enzyme was performed using HEX tool. The grid was fixed at 0.6 and the default FFT (Fast Fourier Transformation) mode was chosen. The algorithm exhaustively searches the entire rotational and translational space of the ligand with respect to the receptors. The ATP binding with the enzyme was obtained as energy value -228.72.Then the energy minimization of the only model and the model complex with ATP was computed by Chimera tool [18] show the potential energy as -13981 .757 and -15605.920 respectively that indicates the ligand ATP is lodge in its proper binding site. From the docked structure the residues that facilitates for binding of the above ligands



CastP server that also showing the pocket 3 having residues 110-CB –ILE 110-O-ILE, 113-CB-ARG, 296-CD2-LEU are responsible for binding (Fig.6). Cross verified result by EMBOSS tool show that the prosite motif of the sequence is from residue 110-113(AGRR). CONCLUSION Acetyl Co-enzyme carboxyl transferase is the main enzyme which catalyses the first step of the lipid synthesis in algae Cyanidium caldarium. The storage lipids in the algae correspond to the biofuel producing capability. In the present work a homology based modeling for the above enzyme has been constructed using MODELLER software. And after various types of model validation software the result suggest that the model is reliable. The stable structure is further subjected to Docking with ATP the key ligand of the enzyme action. The docking result shows that almost similar part of the enzyme is responsible for binding with both the ligand also the residue. The predicted pocket result of the enzyme obtained from Cast P server was found is consistent with the predicted result by EMBOSS tool for the conserved motif. The work basically relates to the in-silico analysis of the fatty acid synthetic pathway leading to accumulation of oils and fats and an understanding about how the pathway could be utilized for the commercial production of algal biofuels. ACKNOWLEDGEMENT We are thankful to CEO, Director and Dean of Majhighariani Institute of Technology & Science,

Ryagada to provide us the MIRC lab for computing facility. REFERENCES 1. Edward M. et al., Nature, 454, 841-845, 2008.

2. Masamichi Akimoto et al., Journal of chemical Engineering of Japan, 28, 349-352, 1995.

3. Qiang Hu et al., The Plant Journal, 54, 621–639, 2008. 4. Van Hannen EJ et al., Eur J Phycol, 37,203-208, 2002. 5. T. Andrew Binkowski et al., Nucleic Acids Res., 31, 3352–3355, 2003. 6. Rice P et al., Trends in Genetics, 16, 276-277, 2000. 7. GloecknerG., J. Mol. Evol., 51, 382-390, 2000. 8. Altschul, S.F. et al ,J. Mol. Biol. 215, 403-410, 1990, 9.A. Sali & T.L. Blundell, J. Mol. Biol., 234,779-815, 1993. 10. Eswar N et al., Mol. Biol, 426,145-159, 2008. 11. Georgii G. Krivov, Proteins Structure, Function, and Bioinformatics, 77, 778-795, 2009. 12. Laskowski R et al, J. Appl. Cryst., 26, 283-291, 1993. 13 V C. Colovos & T. O. Yeats, Protein Sci, 2, 1511-1519, 1993. 14. www.pymol.org. 15. D.W. Ritchie, PROTEINS. Structure, Function and Genetics., 52, 98-106, 2003. 16. L. Mavridis & D.W. Ritchie, J. Chem. Inf. Model., 47, 1787-1796, 2008. 17. Bilder P, et al., Biochemistry, 45, 1712-1722, 2006. 18. Pettersen EF et al., J Comput Chem., 25, 1605-1612, 2004.

Table - 1 Structural information of the Modeled ACCase enzyme


Structural features Information

No. of residues 324

No. of atoms 2572

No. of Hydrogen bonds 225

No. of helices 17

No. of strands 15

No. of turns 28


Table - 2 Ramachandaran plot calculation on 3D model of ACCase enzyme computed with PROCHECK program

% residues in favorable regions 92.3

% residues in additional residue regions 5.2

% residues in generously regions 1.7

% residues in disallowed regions 0.7

% of non Proline and non Glycine residues 100

Figure - 1

Working principle of MODELLER (Sali and Blundell 1993)

Figure - 2

Rasmol view of final 3D structure of the ACCase. Yellow color indicates beta sheets and Red one is the alpha helix.

_____________________________________________________________________________________________________________________ Journal of Natural Science and Technology - Life Sciences and Bioinformatics, 2010, Vol. 2: Page No. 147-152 -150-


Figure - 3

Comparative DOPE value of template and model obtained from MODELLER output

Figure - 4

ERRAT structural quality

Figure - 5

The Ramachandran’s plot for the model protein

_____________________________________________________________________________________________________________________ Journal of Natural Science and Technology - Life Sciences and Bioinformatics, 2010, Vol. 2: Page No. 147-152 -151-


Figure – 6

The docked structure of ATP (Red color) in the pocket residues of the enzyme. (Shown in Blue color)

Citation: Raghunath Satpathy et al., nst Life Sciences and Bioinformatics Vol. 2: 147-152 (2010) License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes, provided the original author and source are credited.


insilico docking

Documents