Cell Biochemistry and Biophysics

, Volume 67, Issue 2, pp 623–633

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

  • Balu K.
  • Vidya Rajendran
  • Rao Sethumadhavan
  • Rituraj Purohit
Original Paper


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.


Cell signalling Flexibility Binding affinity Interactions Hydrogen bonds Molecular dynamics 



Breast cancer anti-oestrogen resistance protein 1


Buried surface area


Cysteine-rich receptor like kinases


Focal adhesion target


Radius of gyration


Solvent accessible surface area

Supplementary material

12013_2013_9551_MOESM1_ESM.tif (313 kb)
Supplementary material 1 (TIFF 313 kb)


  1. 1.
    Kholodenko, B. N. (2006). Cell signalling dynamics in time and space. Nature Reviews Molecular Cell Biology, 7, 165–176.PubMedCrossRefGoogle Scholar
  2. 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.CrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.CrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.CrossRefGoogle Scholar
  16. 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.Google Scholar
  17. 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.CrossRefGoogle Scholar
  18. 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.PubMedCrossRefGoogle Scholar
  19. 19.
    Kumar, A., & Purohit, R. (2012). Computational investigation of pathogenic nsSNPs in CEP63 protein. Gene, 503, 75–82.PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 21.
    Balu, K., & Rituraj, P. (2013). Mutational analysis of TYR gene and its structural consequences in OCA1A. Gene, 513, 184–195.CrossRefGoogle Scholar
  22. 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.CrossRefGoogle Scholar
  23. 23.
    Kumar, A., & Purohit, R. (2012). Computational centrosomics: An approach to understand the dynamic behaviour of centrosome. Gene, 511, 125–126.PubMedCrossRefGoogle Scholar
  24. 24.
    Berman, H. M., Westbrook, J., Feng, Z., et al. (2000). The protein data bank. Nucleic Acids Research, 28, 235–242.PubMedCrossRefGoogle Scholar
  25. 25.
    Kaplan, W., & Littlejohn, T. G. (2001). Swiss-PDB viewer (deep view). Briefings in Bioinformatics, 2, 195–197.PubMedCrossRefGoogle Scholar
  26. 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.CrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 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.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 33.
    McDonald, I. K., & Thornton, J. M. (1994). Satisfying hydrogen bonding potential in proteins. Journal of Molecular Biology, 238, 777–793.PubMedCrossRefGoogle Scholar
  34. 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.CrossRefGoogle Scholar
  35. 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.CrossRefGoogle Scholar
  36. 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. 37.
    Amadei, A., Linssen, A. B., & Berendsen, H. J. C. (1993). Essential dynamics of proteins. Proteins, 17, 412–425.PubMedCrossRefGoogle Scholar
  38. 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.PubMedCrossRefGoogle Scholar
  39. 39.
    Janin, J., Henrick, K., Moult, J., et al. (2003). CAPRI: A critical assessment of predicted interactions. Proteins, 52, 2–9.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 43.
    Fersht, A. R. (1984). Basis of biological specificity. Trends in Biochemical Sciences, 9, 145–147.CrossRefGoogle Scholar
  44. 44.
    Honig, B., & Yang, A. S. (1995). Free energy balance in protein folding. Advances in Protein Chemistry, 46, 27–58.PubMedCrossRefGoogle Scholar
  45. 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.PubMedCrossRefGoogle Scholar
  46. 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.PubMedCrossRefGoogle Scholar
  47. 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. 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

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Balu K.
    • 1
  • Vidya Rajendran
    • 1
  • Rao Sethumadhavan
    • 1
  • Rituraj Purohit
    • 1
    • 2
  1. 1.Bioinformatics Division, School of Bio Sciences and TechnologyVellore Institute of Technology UniversityVelloreIndia
  2. 2.Human Genetics FoundationTorinoItaly

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