Applied Biochemistry and Biotechnology

, Volume 187, Issue 4, pp 1448–1459 | Cite as

In Silico Structure-Based Identification and Validation of Key Residues of Vip3Aa Involving in Lepidopteran Brush Border Receptor Binding

  • Baoyan Chi
  • Haitao Li
  • Jinbo Zhang
  • Panpan Wei
  • Jiguo GaoEmail author
  • Rongmei LiuEmail author


The vegetative insecticidal proteins (VIPs) of Bacillus thuringiensis (Bt) have a broad-spectrum insecticidal activity against Lepidopteran pests and no cross-resistance with the insecticidal crystal protein Cry protein. So there are great potentials for the control of agricultural pests and the resolution of resistance problems. The structural information of Vip3Aa protein and the predicted key amino acid sites on the C-terminal domain of Vip3Aa were analyzed with the methods of bioinformatics such as homology modeling and molecular docking. Site-directed mutagenesis was used to replace these amino acids with alanine, and there was difference in the activities of the mutant protein and Vip3Aa protein. Y619A had improved insecticidal activity against Helicoverpa armigera, but the toxicity of W552A and E627A to Helicoverpa armigera was significantly reduced. The mutants of W552A and E627A had reduced insecticidal activity against Spodoptera exigua. This study demonstrated that the C-terminal domain played an important role in the function of Vip3Aa protein toxin, and the deletion of the side chain of key residues had a significant effect on the activity of the insecticidal protein. This study provides the theoretical basis for revealing the relationship between the structure and function of Vip3Aa protein.


Bacillus thuringiensis (Bt) Vip3A Receptor-binding sites Carbohydrate-binding domain Molecular docking 


Compliance with Ethical Standards

Conflict of Interest

The authors declared that they have no conflict of interest.

Supplementary material

12010_2018_2880_MOESM1_ESM.docx (980 kb)
ESM 1 (DOCX 980 kb)


  1. 1.
    Chakroun, M., Banyuls, N., Bel, Y., Escriche, B., & Ferré, J. (2016). Correction for Chakroun et al., Bacterial vegetative insecticidal proteins (Vip) from Entomopathogenic Bacteria. Microbiology and Molecular Biology Reviews: MMBR, 80(2), 329–350.CrossRefGoogle Scholar
  2. 2.
    Escudero, I. R. D., et al. (2014). A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests. Journal of Invertebrate Pathology, 117(2), 51–55.CrossRefGoogle Scholar
  3. 3.
    Liu, M., Liu, R., Luo, G., Li, H., & Gao, J. (2017). Effects of site-mutations within the 22 kDa no-core fragment of the Vip3Aa11 insecticidal toxin of bacillus thuringiensis. Current Microbiology, 74(5), 655–659.CrossRefGoogle Scholar
  4. 4.
    Selvapandiyan, A., Arora, N., Rajagopal, R., Jalali, S. K., Venkatesan, T., Singh, S. P., & Bhatnagar, R. K. (2001). Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis. Applied and Environmental Microbiology, 67(12), 5855–5858.CrossRefGoogle Scholar
  5. 5.
    Lee, M. K., Curtiss, A., Alcantara, E., & Dean, D. H. (1996). Synergistic effect of the Bacillus thuringiensis toxins CryIAa and CryIAc on the gypsy moth, Lymantria dispar. Applied and Environmental Microbiology, 62(2), 583–586.Google Scholar
  6. 6.
    Gayen, S., Hossain, M. A., & Sen, S. K. (2012). Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis. Journal of Plant Biochemistry and Biotechnology, 21(1), 128–135.CrossRefGoogle Scholar
  7. 7.
    Abdelkefi-Mesrati, L., Boukedi, H., Dammak-Karray, M., Sellami-Boudawara, T., Jaoua, S., & Tounsi, S. (2011). Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. Journal of Invertebrate Pathology, 106(2), 250–254.CrossRefGoogle Scholar
  8. 8.
    Ben, H. D., et al. (2013). Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin. Journal of Invertebrate Pathology, 114(2), 139–143.CrossRefGoogle Scholar
  9. 9.
    Abdelkefimesrati, L., et al. (2011). Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin. Journal of Invertebrate Pathology, 107(3), 198–201.CrossRefGoogle Scholar
  10. 10.
    Chakroun, M., & Ferré, J. (2014). In vivo and in vitro binding of Vip3Aa to Spodoptera frugiperda midgut and characterization of binding sites by (125)I radiolabeling. Applied and Environmental Microbiology, 80(20), 6258–6265.CrossRefGoogle Scholar
  11. 11.
    Palma, L., Escudero, I. D., & Maeztu, M. (2013). Screening of vip genes from a Spanish Bacillus thuringiensis collection and characterization of two Vip3 proteins highly toxic to five lepidopteran crop pests. Biological Control, 66(3), 141–149.CrossRefGoogle Scholar
  12. 12.
    Chi, B., et al. (2017). Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests. Biocontrol Science and Technology, 27(12), 1363–1372.Google Scholar
  13. 13.
    Mushtaq, R., Shakoori, A. R., & Juratfuentes, J. L. (2018). Domain III of Cry1Ac is critical to binding and toxicity against soybean looper (Chrysodeixis includens) but not to Velvetbean Caterpillar (Anticarsia gemmatalis). Toxins, 10(3), 95.CrossRefGoogle Scholar
  14. 14.
    Aftab, A., et al. (2015). In-Silico determination of insecticidal potential ofVip3Aa-Cry1Ac fusion protein against lepidopteran targets using molecular docking. Frontiers in Plant Science, 6, 1–29.Google Scholar
  15. 15.
    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6), 845–858.CrossRefGoogle Scholar
  16. 16.
    Song, F., Chen, C., Wu, S., Shao, E., Li, M., Guan, X., & Huang, Z. (2016). Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation. Scientific Reports, 6(1), 23861.CrossRefGoogle Scholar
  17. 17.
    Pierce, B. G., Wiehe, K., Hwang, H., Kim, B. H., Vreven, T., & Weng, Z. (2014). ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics, 30(12), 1771–1773.CrossRefGoogle Scholar
  18. 18.
    Bett, B., Gollasch, S., Moore, A., James, W., Armstrong, J., Walsh, T., Harding, R., & Higgins, T. J. V. (2017). Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus thuringiensis Vip 3Ba protein are protected against the Maruca pod borer ( Maruca vitrata ). Plant Cell, Tissue and Organ Culture, 131(2), 335–345.CrossRefGoogle Scholar
  19. 19.
    Chen, W. B., Lu, G. Q., Cheng, H. M., Liu, C. X., Xiao, Y. T., Xu, C., Shen, Z. C., & Wu, K. M. (2017). Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests. Journal of Invertebrate Pathology, 149, 59–65.CrossRefGoogle Scholar
  20. 20.
    Bhalla, R., Dalal, M., Panguluri, S. K., Jagadish, B., Mandaokar, A. D., Singh, A. K., & Kumar, P. A. (2005). Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis. FEMS Microbiology Letters, 243(2), 467–472.CrossRefGoogle Scholar
  21. 21.
    Estruch, J. J., Warren, G. W., Mullins, M. A., Nye, G. J., Craig, J. A., & Koziel, M. G. (1996). Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proceedings of the National Academy of Sciences of the United States of America, 93(11), 5389–5394.CrossRefGoogle Scholar
  22. 22.
    Dong, F., Zhang, S., Shi, R., Yi, S., Xu, F., & Liu, Z. (2012). Ser-substituted mutations of Cys residues in bacillus thuringiensis Vip3Aa7 exert a negative effect on its insecticidal activity. Current Microbiology, 65(5), 583–588.CrossRefGoogle Scholar
  23. 23.
    Zhang, J., et al. (2017). Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease. International Journal of Biological Macromolecules, 107, 1220–1226.Google Scholar
  24. 24.
    Lee, M. K., Young, B. A., & Dean, D. H. (1995). Domain III exchanges of Bacillus thuringiensis CryIA toxins affect binding to different gypsy moth midgut receptors. Biochemical and Biophysical Research Communications, 216(1), 306–312.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Northeast Agricultural UniversityHarbinPeople’s Republic of China

Personalised recommendations