Skip to main content
SpringerLink
Log in

Investigation of Binding Phenomenon of NSP3 and p130Cas Mutants and Their Effect on Cell Signalling

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3L469R, NSP3L623E, NSP3R627E) and p130Cas (p130CasF794R, p130CasL787E, p130CasD797R) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3L469R and p130CasD797R mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3L469R and p130CasD797R showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Abbreviations

BCAR1:

Breast cancer anti-oestrogen resistance protein 1

BSA:

Buried surface area

Crk:

Cysteine-rich receptor like kinases

FAT:

Focal adhesion target

Rg:

Radius of gyration

SASA:

Solvent accessible surface area

References

  1. Kholodenko, B. N. (2006). Cell signalling dynamics in time and space. Nature Reviews Molecular Cell Biology, 7, 165–176.

    Article  PubMed  CAS  Google Scholar 

  2. Mace, P. D., Wallez, Y., Dobaczewska, M. K., et al. (2011). NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling. Nature Structural & Molecular Biology, 18, 1381–1387.

    Article  CAS  Google Scholar 

  3. Bargon, S. D., Gunning, P. W., & O’Neill, G. M. (2005). The Cas family docking protein, HEF1, promotes the formation of neurite-like membrane extensions. Biochimica et Biophysica Acta, 1746, 143–154.

    Article  PubMed  CAS  Google Scholar 

  4. Hauck, C. R., Hsia, D. A., Puente, X. S., Cheresh, D. A., & Schlaepfer, D. D. (2002). FRNK blocks v-Src-stimulated invasion and experimental metastases without effects on cell motility or growth. The EMBO Journal, 21, 6289–6302.

    Article  PubMed  CAS  Google Scholar 

  5. Brábek, J., Constancio, S. S., Siesser, P. F., et al. (2005). Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells. Molecular Cancer Research, 3, 307–315.

    Article  PubMed  Google Scholar 

  6. Sakakibara, A., Ohba, Y., Kurokawa, K., Matsuda, M., & Hattori, S. (2002). Novel function of chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation. Journal of Cell Science, 115, 4915–4924.

    Article  PubMed  CAS  Google Scholar 

  7. Honda, H., Nakamoto, T., Sakai, R., & Hirai, R. (1999). p130(Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochemical and Biophysical Research Communications, 262, 25–30.

    Article  PubMed  CAS  Google Scholar 

  8. Garron, M. L., Arsenieva, D., Zhong, J., et al. (2009). Structural insights into the association between BCAR3 and Cas family members, an atypical complex implicated in anti-oestrogen resistance. Journal of Molecular Biology, 386, 190–203.

    Article  PubMed  CAS  Google Scholar 

  9. Al-Shami, A., Wilkins, C., Crisostomo, J., Seshasayee, D., Martin, F., Xu, N., et al. (2010). The adaptor protein sh2d3c is critical for marginal zone B cell development and function. Journal of Immunology, 185, 327–334.

    Article  CAS  Google Scholar 

  10. Tikhmyanova, N., Little, J. L., & Golemis, E. A. (2010). CAS proteins in normal and pathological cell growth control. Cellular and Molecular Life Sciences, 67, 1025–1048.

    Article  PubMed  CAS  Google Scholar 

  11. Browne, C. D., Hoefer, M. M., Chintalapati, S. K., et al. (2010). SHEP1 partners with CasL to promote marginal zone B-cell maturation. Proceedings of the National Academy of Sciences of the United States of America, 107, 18944–18949.

    Article  PubMed  CAS  Google Scholar 

  12. Borre, P. V., Near, R. I., Makkinje, A., Mostoslavsky, G., & Lerner, A. (2011). BCAR3/AND-34 can signal independent of complex formation with CAS family members of the presence of p130Cas. Cell Signalling, 23, 1030–1040.

    Article  Google Scholar 

  13. Klemke, R. L., Leng, J., Molander, R., et al. (1998). CAS/Crk coupling serves as a ‘molecular switch” for induction of cell migration. Journal of Cell Biology, 140, 961–972.

    Article  PubMed  CAS  Google Scholar 

  14. Rajendran, V., Purohit, R., & Sethumadhavan, R. (2012). In silico investigation of molecular mechanism of laminopathy cause by a point mutation (R482 W) in lamin A/C protein. Amino Acids, 43, 603–615.

    Article  PubMed  CAS  Google Scholar 

  15. Purohit, R., Rajendran, V., & Sethumadhavan, R. (2011). Studies on adaptability of binding residues and flap region of TMC-114 resistance HIV-1 protease mutants. Journal of Biomolecular Structure & Dynamics, 29, 137–152.

    Article  CAS  Google Scholar 

  16. Purohit, R., & Sethumadhavan, R. (2009). Structural basis for the resilience of Darunavir (TMC114) resistance major flap mutations of HIV-1 protease. Interdisciplinary Reviews, 1, 320–328.

    CAS  Google Scholar 

  17. Purohit, R., Rajendran, V., & Sethumadhavan, R. (2011). Relationship between mutation of serine residue at 315th position in M. tuberculosis catalase-peroxidase enzyme and isoniazid susceptibility: An in silico analysis. Journal of Molecular Modelling, 17, 869–877.

    Article  CAS  Google Scholar 

  18. Purohit, R., Rajasekaran, R., Sudandiradoss, C., et al. (2008). Studies on flexibility and binding affinity of Asp25 of HIV-1 protease mutants. International Journal of Biological Macromolecules, 42, 386–391.

    Article  PubMed  CAS  Google Scholar 

  19. Kumar, A., & Purohit, R. (2012). Computational investigation of pathogenic nsSNPs in CEP63 protein. Gene, 503, 75–82.

    Article  PubMed  CAS  Google Scholar 

  20. Kumar, A., & Purohit, R. (2012). Computational screening and molecular dynamics simulation of disease associated nsSNPs in CENP-E. Mutation Research, 738–739, 28–37.

    Article  PubMed  Google Scholar 

  21. Balu, K., & Rituraj, P. (2013). Mutational analysis of TYR gene and its structural consequences in OCA1A. Gene, 513, 184–195.

    Article  Google Scholar 

  22. Kumar, A., Rajendran, V., Sethumadhavan, R., & Purohit, R. (2012). In silico prediction of a disease-associated STIL mutant and its affect on the recruitment of centromere protein J (CENPJ). FEBS Open Biology, 2, 285–293.

    Article  Google Scholar 

  23. Kumar, A., & Purohit, R. (2012). Computational centrosomics: An approach to understand the dynamic behaviour of centrosome. Gene, 511, 125–126.

    Article  PubMed  CAS  Google Scholar 

  24. Berman, H. M., Westbrook, J., Feng, Z., et al. (2000). The protein data bank. Nucleic Acids Research, 28, 235–242.

    Article  PubMed  CAS  Google Scholar 

  25. Kaplan, W., & Littlejohn, T. G. (2001). Swiss-PDB viewer (deep view). Briefings in Bioinformatics, 2, 195–197.

    Article  PubMed  CAS  Google Scholar 

  26. Hess, B., Kutzner, C., Spoel, D. V. D., & Lindahl, E. (2008). GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. Journal of Chemical Theory and Computation, 4, 435–447.

    Article  CAS  Google Scholar 

  27. Dominguez, C., Boelens, R., & Bonvin, A. M. (2003). HADDOCK: A protein–protein docking approach based on biochemical or biophysical information. Journal of the American Chemical Society, 125, 1731–1737.

    Article  PubMed  CAS  Google Scholar 

  28. de Vries, S. J., van Dijk, M., & Bonvin, A. M. J. J. (2010). The HADDOCK web server for data driven biomolecular docking. Nature Protocols, 5, 883–897.

    Article  PubMed  Google Scholar 

  29. Nilges, M. (1995). Calculation of protein structures with ambiguous distance restraints. Automated assignment of ambiguous NOE crosspeaks and disulphide connectivities. Journal of Molecular Biology, 245, 645–660.

    Article  PubMed  CAS  Google Scholar 

  30. Nilges, M., Macias, M. J., O’Donoghue, S. I., & Oschkinat, H. (1997). Automated NOESY interpretation with ambiguous distance restraints: The refined NMR solution structure of the pleckstrin homology domain from beta-spectrin. Journal of Molecular Biology, 269, 408–422.

    Article  PubMed  CAS  Google Scholar 

  31. Brunger, A. T., Adams, P. D., Clore, G. M., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallographica. Section D, Biological Crystallography, 54, 905–921.

    Article  PubMed  CAS  Google Scholar 

  32. Wallace, A. C., Laskowski, R. A., & Thornton, J. M. (1995). LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Protein Engineering, 8, 127–134.

    Article  PubMed  CAS  Google Scholar 

  33. McDonald, I. K., & Thornton, J. M. (1994). Satisfying hydrogen bonding potential in proteins. Journal of Molecular Biology, 238, 777–793.

    Article  PubMed  CAS  Google Scholar 

  34. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., et al. (1984). Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics, 8, 3684–3690.

    Article  Google Scholar 

  35. Cheatham, T. E, I. I. I., Miller, J. L., Fox, T., et al. (1995). Molecular dynamics simulations on solvated biomolecular systems: The particle mesh Ewald method leads to stable trajectories of DNA, RNA, and proteins. Journal of the American Chemical Society, 14, 4193–4194.

    Article  Google Scholar 

  36. Turner, P. J. (2005). XMGRACE, version 5. 1. 19. Beaverton, OR: Center for Coastal and Land-Margin Research, Oregon Graduate Institute of Science and Technology.

    Google Scholar 

  37. Amadei, A., Linssen, A. B., & Berendsen, H. J. C. (1993). Essential dynamics of proteins. Proteins, 17, 412–425.

    Article  PubMed  CAS  Google Scholar 

  38. Halperin, I., Ma, B., Wolfson, H., & Nussinov, R. (2002). Principle of docking: An overview of search algorithms and a guide to scoring functions. Proteins, 47, 409–443.

    Article  PubMed  CAS  Google Scholar 

  39. Janin, J., Henrick, K., Moult, J., et al. (2003). CAPRI: A critical assessment of predicted interactions. Proteins, 52, 2–9.

    Article  PubMed  CAS  Google Scholar 

  40. Dijk, M. V., Dijk, A. D. V., Hsu, V., et al. (2006). Information-driven protein-DNA docking using HADDOCK: It is a matter of flexibility. Nucleic Acids Research, 34, 3317–3325.

    Article  PubMed  Google Scholar 

  41. Teng, S., Madej, T., Panchenko, A., & Alexov, E. (2009). Modelling effects of human single nucleotide polymorphisms on protein–protein interactions. Biophysical Journal, 96, 2178–2188.

    Article  PubMed  CAS  Google Scholar 

  42. Zhang, Z., Norris, J., Schwartz, C., & Alexov, E. (2011). In silico and in vitro investigations of the mutability of disease-causing missense mutation sites in spermine synthase. PLoS ONE, 6(5), e20373.

    Article  PubMed  CAS  Google Scholar 

  43. Fersht, A. R. (1984). Basis of biological specificity. Trends in Biochemical Sciences, 9, 145–147.

    Article  Google Scholar 

  44. Honig, B., & Yang, A. S. (1995). Free energy balance in protein folding. Advances in Protein Chemistry, 46, 27–58.

    Article  PubMed  CAS  Google Scholar 

  45. Bartlett, P. A., & Marlowe, C. K. (1987). Evaluation of the intrinsic binding energy from hydrogen bonding group in an enzyme inhibitor. Science, 235, 569–571.

    Article  PubMed  CAS  Google Scholar 

  46. Gao, J., Mammen, M., & Whitesides, G. M. (1996). Evaluating electrostatic contributions to binding with the use of protein charge ladders. Science, 272, 535–537.

    Article  PubMed  CAS  Google Scholar 

  47. Kumar, A., Rajendran, V., Sethumadhavan, R., & Purohit, R. (2013). Computational investigation of cancer-associated molecular mechanism in Aurora A (S155R) mutation. Cell Biochemistry and Biophysics,. doi:10.1007/s12013-013-9524-9.

    Google Scholar 

  48. Rajendran, V., & Sethumadhavan, R. (2013). Drug resistance mechanism of PncA in mycobacterium tuberculosis. Journal of Biomolecular Structure & Dynamics,. doi:10.1080/07391102.2012.759885.

    Google Scholar 

Download references

Acknowledgments

Authors gratefully acknowledge the management of Vellore Institute of Technology University for providing the facilities to carry out this work. We thank the anonymous reviewers for their helpful comments and critical reading of the manuscript.

Conflict of interest

  Authors have no potential conflict of interest to disclose.

Author information

Authors and Affiliations

  1. Bioinformatics Division, School of Bio Sciences and Technology, Vellore Institute of Technology University, Vellore, 632014, Tamil Nadu, India

    Balu K., Vidya Rajendran, Rao Sethumadhavan & Rituraj Purohit

  2. Human Genetics Foundation, via Nizza 52, 10126, Torino, Italy

    Rituraj Purohit

Authors
  1. Balu K.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vidya Rajendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rao Sethumadhavan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rituraj Purohit
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rituraj Purohit.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (TIFF 313 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Balu K., Rajendran, V., Sethumadhavan, R. et al. Investigation of Binding Phenomenon of NSP3 and p130Cas Mutants and Their Effect on Cell Signalling. Cell Biochem Biophys 67, 623–633 (2013). https://doi.org/10.1007/s12013-013-9551-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-013-9551-6

Keywords

Search

Navigation