Advertisement

Archives of Virology

, 152:847 | Cite as

Structure of the extended diarrhea-inducing domain of rotavirus enterotoxigenic protein NSP4

  • R. Deepa
  • C. Durga Rao
  • K. Suguna
Article

Summary.

Rotavirus nonstructural protein 4 (NSP4) is a multidomainal and multifunctional protein and is recognized as the first virus-encoded enterotoxin. Extensive efforts to crystallize the complete cytoplasmic tail (CT), which exhibits all the known biological functions, have been unsuccessful, and to date, the structure of only a synthetic peptide corresponding to amino acids (aa) 95–137 has been reported. Recent studies indicate that the interspecies-variable domain (ISVD) from aa 135 to 141 as well as the extreme C-terminus are critical determinants of virus virulence and the diarrhea-inducing ability of the protein. Among the five NSP4 genotypes identified, those belonging to genotypes A1, B and C possess either a proline at position 138 or a glycine at 140, while those of A2, D and E lack these residues in the ISVD, suggesting conformational differences in this region among different NSP4s. Here, we examined the crystallization properties of several deletion mutants and report the structure of a recombinant mutant, NSP4:95–146, lacking the N-terminal 94 and C-terminal 29 aa, from SA11 (A1) and I321 (A2) at 1.67 and 2.7 Å respectively. In spite of the high resolution of one of the structures, electron density for the C-terminal 9 residues could not be seen for either of the mutants, and the crystal packing resulted in the creation of a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as the crystal packing owing to the additional space required to accommodate the un structured virulence-determining region. The crystal structure and secondary structure prediction of the NSP4:95–146 mutants from different genotypes suggest that the region C-terminal to aa 137 in all the NSP4 proteins is likely to be unstructured, and this might be of structural and biological functional significance.

Keywords

Coiled Coil Helix Axis NSP4 Gene NSP4 Protein Potassium Sodium Tartarate Tetrahydrate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Angel, J, Tang, B, Feng, N, Greenberg, HB, Bass, D 1998Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrheaJ Infect Dis177455458CrossRefPubMedGoogle Scholar
  2. Au, KS, Chan, WK, Burns, JW, Estes, MK 1989Receptor activity of rotavirus nonstructural glycoprotein NS28J Virol6345534562PubMedGoogle Scholar
  3. Ball, JM, Tian, P, Zeng, CQY, Morris, AP, Estes, MK 1996Age-dependent diarrhea induced by a rotavirus nonstructural glycoproteinScience272101104CrossRefPubMedGoogle Scholar
  4. Boshuizen, JA, Rossen, JW, Sitaram, CK, Kimenai, FF, Simons-Oosterhuis, Y, Laffeber, C, Buller, HA, Einerhand, AW 2004Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectinJ Virol781004510053CrossRefPubMedGoogle Scholar
  5. Bowman, GD, Nodelman, NM, Levy, O, Lin, SL, Tian, P, Zamb, TJ, Udem, SA, Venkataraghavan, B, Schutt, CE 2000Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding siteJ Mol Biol304861871CrossRefPubMedGoogle Scholar
  6. Brunger, AT, Adams, PD, Rice, JM 1998Recent developments for the efficient crystallographic refinement of macromolecular structuresCurr Opin Struct Biol8606611CrossRefPubMedGoogle Scholar
  7. Chan, WK, Au, KS, Estes, MK 1988Topology of the simian rotavirus nonstructural glycoproteins (NS28) in the endoplasmic reticulum membraneVirology164435442CrossRefPubMedGoogle Scholar
  8. Chang, KO, Kim, YJ, Saif, LJ 1999Comparisons of nucleotide and deduced amino acid sequences of NSP4 genes of virulent and attenuated pairs of group A and C rotavirusesVirus Genes18229233CrossRefPubMedGoogle Scholar
  9. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Cryst D50: 760-63Google Scholar
  10. Deepa, R, Jagannath, MR, Kesavulu, MM, Durga Rao, C, Suguna, K 2004Expression, purification, crystallization and preliminary crystallographic analysis of the diarrhoea-causing and virulence-determining region of rotaviral nonstructural protein NSP4Acta CrystD60135136Google Scholar
  11. DeLano, WL 2002The PyMOL Molecular Graphics SystemDeLano Scientific, San CarlosCA, USAGoogle Scholar
  12. Estes, MK 2001Rotaviruses and their replicationKnipe, DMHowley, PMGriffin, DELamb, RAMartin, MARoizman, BStraus, SE eds. Fields virology4Lippincott Williams and WilkinsPhiladelphia17471785Google Scholar
  13. Isupov, MN, Fleming, TM, Dalby, AR, Crowhurst, GS, Bourne, PC, Littlechild, JA 1999Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricusJ Mol Biol291651660CrossRefPubMedGoogle Scholar
  14. Jagannath, MR, Kesavulu, MM, Deepa, R, Sastri, PN, Kumar, SS, Suguna, K, Rao, CD 2006N- and C-terminal cooperation in rotavirus enterotoxin: novel mechanism of modulation of the properties of a multifunctional protein by a structurally and functionally overlapping conformational domainJ Virol80412425CrossRefPubMedGoogle Scholar
  15. Jagannath, MR, Vethanayagam, RR, Reddy, BSY, Raman, S, Rao, CD 2000Characterization of human symptomatic rotavirus isolates MP409 and MP480 having “long” RNA electropherotype and subgroup I specificity, highly related to the P6[1], G8 type bovine rotavirus A5, from Mysore, IndiaArch Virol14513391357CrossRefPubMedGoogle Scholar
  16. Jancarik, J, Kim, SH 1991Sparse matrix sampling: a screening method for crystallization of proteinsJ Appl Cryst24409411CrossRefGoogle Scholar
  17. Johansen, K, Hinkula, J, Espinoza, F, Levi, M, Zeng, C, Ruden, U, Vesikari, T, Estes, MK, Svennson, L 1999Humoral and cell-mediated immune response in humans to the NSP4 enterotoxin of rotavirusJ Med Virol59366371CrossRefGoogle Scholar
  18. Jones, TA, Zou, JY, Cowan, SW, Kjeldgaard, M 1991Improved methods for building protein models in electron density maps and the location of errors in these modelsActa CrystA47110119Google Scholar
  19. Laskowski, RA, MacArthur, MW, Moss, DS, Thornton, JM 1993PROCHECK — a program to check the stereochemical quality of protein structuresJ Appl Cryst26283291CrossRefGoogle Scholar
  20. Lin, SL, Tian, P 2003Detailed computational analysis of a comprehensive set of group A rotavirus NSP4 proteinsVirus Genes26271282CrossRefPubMedGoogle Scholar
  21. Maass, DR, Atkinson, PH 1990Rotavirus proteins VP7, NS28, and VP4 form oligomeric structuresJ Virol6426322641PubMedGoogle Scholar
  22. Manikandan, K, Ramakumar, S 2004The occurrence of C–H…O hydrogen bonds in alpha-helices and helix termini in globular proteinsProteins56768781CrossRefPubMedGoogle Scholar
  23. Meyer, JC, Bergmann, CC, Bellamy, AR 1989Interaction of rotavirus cores with the nonstructural glycoprotein NS28Virology17198107CrossRefPubMedGoogle Scholar
  24. Mohan, KV, Dermody, TS, Atreya, CD 2000Mutations selected in rotavirus enterotoxin NSP4 depend on the context of its expressionVirology275125132CrossRefPubMedGoogle Scholar
  25. Murshudov, GN, Vagin, AA, Dodson, EJ 1997Refinement of macromolecular structures by the maximum-likelihood methodActa CrystD53240255Google Scholar
  26. Navaza, J 1994AMoRe — an automated package for molecular replacementActa CrystA50157163Google Scholar
  27. Newton, K, Meyer, JC, Bellamy, AR, Taylor, JA 1997Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cellsJ Virol7194589465PubMedGoogle Scholar
  28. Oka, T, Nakagomi, T, Nakagomi, O 2001A lack of consistent amino acid substitutions in NSP4 between rotaviruses derived from diarrheal and asymptom atically infected kittensMicrobial Immunol45173177Google Scholar
  29. Powell, HR 1999The Rossmann Fourier autoindexing algorithm in MOSFLMActa CrystD5516901695Google Scholar
  30. Roberts, L 2004Vaccines. Rotavirus vaccine: second chanceScience30518901893CrossRefPubMedGoogle Scholar
  31. Schagger, H, von Jagaw, G 1987Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDaAnal Biochem166368379CrossRefPubMedGoogle Scholar
  32. Silvestri, LS, Tortorici, MA, Vasquez-Del Carpio, R, Patton, JT 2005Rotavirus glycoprotein NSP4 is a modulator of viral transcription in the infected cellJ Virol791516515174CrossRefPubMedGoogle Scholar
  33. Taylor, JA, Meyer, JC, Legge, MA, O’Brien, T, Street, JE, Lord, VJ, Bergmann, CC, Bellamy, AR 1992Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methi onine residue is essential for ligand bindingJ Virol6635663572PubMedGoogle Scholar
  34. Taylor, JA, O’Brien, JA, Lord, VJ, Meyer, JC, Bellamy, AR 1993The RER-localized rotavirus intracellular receptor: a truncated purified soluble form is multivalent and binds virus particlesVirology194807814CrossRefPubMedGoogle Scholar
  35. Taylor, JA, O’Brien, JA, Yeager, M 1996The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domain. EMBO J1544694476PubMedGoogle Scholar
  36. Tian, P, Estes, MK, Hu, Y, Ball, JM, Zeng, CQ, Schilling, WP 1995The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulumJ Virol6957635772PubMedGoogle Scholar
  37. Tian, P, Ball, JM, Zeng, CQ, Estes, MK 1996The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activityJ Virol7069736981PubMedGoogle Scholar
  38. Ward, RL, Mason, BB, Bernstein, DI, Sander, DS, Smith, VE, Zandle, GA, Rappaport, RS 1997Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 geneJ Virol7162676270PubMedGoogle Scholar
  39. Winn, MD, Isupov, MN, Murshudov, GN 2001Use of TLS parameters to model anisotropic displacements in macromolecular refinementActa CrystD57122133Google Scholar
  40. Xu, A, Bellamy, AR, Taylor, JA 2000Immobilization of the early secretory pathway by a virus glycoprotein that binds to microtubulesEMBO J1964656474CrossRefPubMedGoogle Scholar
  41. Zhang, M, Zeng, CQ, Dong, Y, Ball, JM, Saif, LJ, Morris, AP, Estes, MK 1998Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulenceJ Virol7236663672PubMedGoogle Scholar
  42. Zhang, M, Zeng, CQ, Morris, AP, Estes, MK 2000A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cellsJ Virol741166311670CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • R. Deepa
    • 1
    • 2
  • C. Durga Rao
    • 1
  • K. Suguna
    • 2
  1. 1.Department of Microbiology and Cell BiologyIndian Institute of ScienceBangaloreIndia
  2. 2.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia

Personalised recommendations