Designing and analyzing the structure of Tat-BoNT/A(1-448) fusion protein: An in silico approach

Document Type : Original article

Authors

1 Applied Microbiology Research center, Baqiyatallah University of Medical Sciences, Tehran, Iran.

2 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

3 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Abstract

Clostridium botulinum type A (BoNT/A) produces a neurotoxin recently found to be useful as an injectable drug for the treatment of abnormal muscle contractions. The catalytic domain of this toxin which is responsible for the main toxin activity is a zinc metalloprotease that inhibits the release of neurotransmitter mediators in neuromuscular junctions. A cell penetrating cationic peptide, Tat, which is a truncated N-terminal part of the Tat protein from human immunodeficiency virus, can help the toxin penetrate the skin uninvasively. This study aimed at an in silico analyses of the Tat-BoNT/A(1-448) fusion protein structure. A genomic construct was designed and optimized based on E. coli codon usage. The structure of mRNA as well as the properties of hypothetical chimeric protein was then analyzed by bioinformatic tools. Afterwards, the secondary and tertiary structures of the fusion protein were predicted by GOR4 and I-TASSER online web servers. The interaction with synaptosomal associated protein 25kDa (SNAP-25) was also analyzed as a natural substrate for the toxin. Based on the studied secondary and tertiary structures of the protein, the selected order of fusion proteins provides the natural activity of each peptide. Energy calculating data show that the acquired thermodynamic ensemble related to the mRNA structure was-1473.2 kJ/mol (-352.10 kcal/mol) and both total protein energy (Etotal) and shape related energy (Eshape) were calculated as -2294.2kJ/mol (-548.32 kcal/mol). The stability index of TAT-BoNT/A was computed to be 27.22 which has an acceptable stability as compared to that of native BoNT/A (22.39). 

Keywords

References

1.Montecucco C, Molgo J (2005) Botulinal neurotoxins: revival of an old killer. Curr Opin Pharmacol 5:274-9
2.Proft T, editor. Microbial Toxins: Current Research and Future Trends: Horizon Scientific Press; 2009.
3.Thanongsaksrikul J, Chaicumpa W (2011) Botulinum neurotoxins and botulism: a novel therapeutic approach. Toxins (Basel) 3:469-88.
4.Yang Y, Xia Z, Liu Y (2000) SNAP-25 functional domains in SNARE core complex assembly and glutamate release of cerebellar granule cells. The Journal of biological chemistry 275:29482-7.
5.Dhaked RK, Singh MK, Singh P, Gupta P (2010) Botulinum toxin: bioweapon & magic drug. Indian J Med Res 132:489-503.
6.Turton K, Chaddock JA, Acharya KR (2002) Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 27:552-8.
7.Munchau A, Bhatia KP (2000) Uses of botulinum toxin injection in medicine today. BMJ 320:161-5.
8.Johnson EA (1999) Clostridial toxins as therapeutic agents: benefits of nature's most toxic proteins. Annu. Rev. Microbiol. 53:551-75.
9.Morris MC, Deshayes S, Heitz F, Divita G (2008) Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biology of the cell / under the auspices of the European Cell Biology Organization 100:201-17.
10. Doolittle ED. Methods in Enzymology,R.F. 1996. p. 540-53.
11. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:W126-31.
12. Puigbo P, Romeu A, Garcia-Vallve S (2008) HEG-DB: a database of predicted highly expressed genes in prokaryotic complete genomes under translational selection. Nucleic Acids Res 36:D524-7.
13. Nazarian S, Mousavi Gargari SL, Rasooli I, Amani J, Bagheri S, Alerasool M (2012) An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. Journal of microbiological methods 90:36-45.
14. Amani J, Mousavi SL, Rafati S, Salmanian AH (2009) In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications. Theor Biol Med Model 6:28.
15. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31:3406-15.
16. Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL (2008) The Vienna RNA websuite. Nucleic Acids Res 36:W70-4.
17. Walker JM. The proteomics protocols handbook. Totowa, N.J.: Humana Press; 2005. xviii, 988 p. p.
18. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725-38.
19. Zhang Y. I-TASSER server for protein 3D structure prediction [Research Support, Non-U.S. Gov't]. 2008 [cited 9]. 2008/01/25:[40]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18215316.
20. Roy A, Yang J, Zhang Y (2012) COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res 40:W471-7.
21. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363-71.
22. Lovell SC, Davis IW, Arendall WB, 3rd, de Bakker PI, Word JM, Prisant MG, et al. (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50:437-50.
23. Smialowski P, Martin-Galiano AJ, Mikolajka A, Girschick T, Holak TA, Frishman D (2007) Protein solubility: sequence based prediction and experimental verification. Bioinformatics 23:2536-42.
24. Ritchie DW (2008) Recent progress and future directions in protein-protein docking. Curr Protein Pept Sci 9:1-15.
25. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40.
26. Asahi M, Rammohan R, Sumii T, Wang X, Pauw RJ, Weissig V, et al. (2003) Antiactin-targeted immunoliposomes ameliorate tissue plasminogen activator-induced hemorrhage after focal embolic stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 23:895-9.
27. Waugh A, Gendron P, Altman R, Brown JW, Case D, Gautheret D, et al. (2002) RNAML: a standard syntax for exchanging RNA information. Rna 8:707-17.
28. Rawat R, Ashraf Ahmed S, Swaminathan S (2008) High level expression of the light chain of botulinum neurotoxin serotype C1 and an efficient HPLC assay to monitor its proteolytic activity. Protein Expr Purif 60:165-9.
29. Mueller J, Kretzschmar I, Volkmer R, Boisguerin P (2008) Comparison of cellular uptake using 22 CPPs in 4 different cell lines. Bioconjug Chem 19:2363-74.
30. Rajagopalan R, Xavier J, Rangaraj N, Rao NM, Gopal V (2007) Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery. J Gene Med 9:275-86.
31. Yesylevskyy S, Marrink SJ, Mark AE (2009) Alternative mechanisms for the interaction of the cell-penetrating peptides penetratin and the TAT peptide with lipid bilayers. Biophys J 97:40-9.
32. Thomas A, Lins L, Divita G, Brasseur R (2010) Realistic modeling approaches of structure-function properties of CPPs in non-covalent complexes. Biochim Biophys Acta 1798:2217-22.

Files

Share

How to cite

Statistics