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The role of the N-terminal loop in the function of the colicin E7 nuclease domain

  • Anikó Czene
  • Eszter Németh
  • István G. Zóka
  • Noémi I. Jakab-Simon
  • Tamás Körtvélyesi
  • Kyosuke Nagata
  • Hans E. M. Christensen
  • Béla Gyurcsik
Original Paper

Abstract

Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX14NX8HX3H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446–576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn2+ ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn2+ ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485–487, 511–515 and 570–571, and in ΔN4-NColE7 those between residues 467–468, 530–535 and 570–571.

Keywords

Metallonuclease Colicin E7 N-terminally truncated mutants Zinc(II) binding 

Notes

Acknowledgments

This work has received support through the Hungarian Science Foundation (OTKA-NKTH CK80850), TÁMOP-4.2.1/B-09/1/KONV-2010-0005 and TÁMOP-4.2.2/B-10/1-2010-0012. The computational resources at High Performance Computing of the University of Szeged and financial support from the European Union Research Infrastructure Action FP7 (Integrated Activity on Synchrotron and Free Electron Laser Science, contract no. FP7/2007-2013; no. 226716) are also gratefully acknowledged. B.G. thanks the Japan Society for the Promotion of Science, and I.N.J-S. and A.C. thank the Hungarian Scholarship Board for the fellowships provided.

Supplementary material

775_2013_975_MOESM1_ESM.pdf (992 kb)
Supplementary material 1 (PDF 993 kb)

References

  1. 1.
    Chak K-F, Kuo W-S, Lu F-M, James R (1991) J Gen Microbiol 137:91–100PubMedGoogle Scholar
  2. 2.
    Lin Y-H, Liao C-C, Liang P-H, Yuan HS, Chak K-F (2004) Biochem Biophys Res Commun 318:81–87PubMedCrossRefGoogle Scholar
  3. 3.
    Liao C-C, Hsia K-C, Liu Y-W, Leng P-H, Yuan HS, Chak K-F (2001) Biochem Biophys Res Commun 284:556–562PubMedCrossRefGoogle Scholar
  4. 4.
    Cheng Y-S, Shi Z, Doudeva LG, Yang W-Z, Chak K-F, Yuan HS (2006) J Mol Biol 356:22–31PubMedCrossRefGoogle Scholar
  5. 5.
    Sui M-J, Tsai L-C, Hsia K-C, Doudeva LG, Ku W-Y, Han GW, Yuan HS (2002) Protein Sci 11:2947–2957PubMedCrossRefGoogle Scholar
  6. 6.
    Cheng Y-S, Hsia K-C, Doudeva LG, Chak K-F, Yuan HS (2002) J Mol Biol 324:227–236PubMedCrossRefGoogle Scholar
  7. 7.
    Chak K-F, Safo MK, Ku W-Y, Hsieh S-Y, Yuan HS (1996) Proc Natl Acad Sci USA 93:6437–6442PubMedCrossRefGoogle Scholar
  8. 8.
    Hsieh S-Y, Ko T-P, Tseng M-Y, Ku W-Y, Chak K-F, Yuan HS (1997) EMBO J 16:1444–1454PubMedCrossRefGoogle Scholar
  9. 9.
    Dennis CA, Videler H, Paupit RA, Wallis R, James R, Moore GR, Kleanthous C (1998) Biochem J 333:183–191PubMedGoogle Scholar
  10. 10.
    Ko T-P, Liao C-C, Ku W-Y, Chak K-F, Yuan HS (1999) Structure 7:91–102PubMedCrossRefGoogle Scholar
  11. 11.
    Kleanthous C, Walker D (2001) Trends Biochem Sci 26:624–631PubMedCrossRefGoogle Scholar
  12. 12.
    Kolade OO, Carr SB, Kühlmann UC, Pommer A, Kleanthous C, Bouchcinsky CA, Hemmings AM (2002) Biochimie 84:439–446PubMedCrossRefGoogle Scholar
  13. 13.
    Orlowski J, Bujnicki JM (2008) Nucleic Acids Res 36:3552–3569PubMedCrossRefGoogle Scholar
  14. 14.
    Eastberg JH, Eklund J, Monnat R, Stoddard BL (2007) Biochemistry 46:7215–7225PubMedCrossRefGoogle Scholar
  15. 15.
    Mehta P, Katta K, Krishnaswamy S (2004) Protein Sci 13:295–300PubMedCrossRefGoogle Scholar
  16. 16.
    Hsia K-C, Chak K-F, Liang P-H, Cheng Y-S, Ku W-Y, Yuan HS (2004) Structure 12:205–214PubMedGoogle Scholar
  17. 17.
    Michel-Briand Y, Baysse C (2002) Biochimie 84:499–510PubMedCrossRefGoogle Scholar
  18. 18.
    Shen BW, Landthaler M, Shub DA, Stoddard BL (2004) J Mol Biol 342:43–56PubMedCrossRefGoogle Scholar
  19. 19.
    Ghosh M, Meiss G, Pingoud A, London RE, Pedersen LC (2005) J Biol Chem 280:27990–27997PubMedCrossRefGoogle Scholar
  20. 20.
    Kriukiene E, Lubiene J, Lagunavicius A, Lubys A (2005) Biochim Biophys Acta 1751:194–204PubMedCrossRefGoogle Scholar
  21. 21.
    Saravanan M, Bujnicki JM, Cymerman IA, Rao DN, Nagaraja V (2004) Nucleic Acids Res 32:6129–6135PubMedCrossRefGoogle Scholar
  22. 22.
    Saravanan M, Vasu K, Ghosh S, Nagaraja V (2007) J Biol Chem 282:32320–32326PubMedCrossRefGoogle Scholar
  23. 23.
    Cymerman IA, Obarska A, Skowronek KJ, Lubys A, Bujnicki MJM (2006) Proteins 65:867–876Google Scholar
  24. 24.
    Jakubauskas A, Giedriene J, Bujnicki JM, Janulaitis A (2007) J Mol Biol 370:157–169PubMedCrossRefGoogle Scholar
  25. 25.
    Sokolowska M, Czapinska H, Bochtler M (2009) Nucleic Acids Res 37:3799–3810PubMedCrossRefGoogle Scholar
  26. 26.
    Veluchamy A, Mary S, Acharya V, Mehta P, Deva T, Krishnaswamy S (2009) Bioinformation 6:80–83CrossRefGoogle Scholar
  27. 27.
    Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer ELL, Bateman A (2008) Nucleic Acids Res 36:D281–D288PubMedCrossRefGoogle Scholar
  28. 28.
    Huang H, Yuan HS (2007) J Mol Biol 368:812–821PubMedCrossRefGoogle Scholar
  29. 29.
    Wang Y-T, Yang W-J, Li C-L, Doudeva LG, Yuan HS (2007) Nucleic Acids Res 35:584–594PubMedCrossRefGoogle Scholar
  30. 30.
    Papadakos G, Wojdyla JA, Kleanthous C (2012) Q Rev Biophys 45:57–103PubMedCrossRefGoogle Scholar
  31. 31.
    Mate MJ, Kleanthous C (2004) J Biol Chem 279:34763–34769PubMedCrossRefGoogle Scholar
  32. 32.
    Doudeva LG, Huang H, Hsia K-C, Shi Z, Li C-L, Shen Y, Cheng C-L, Yuan HS (2006) Protein Sci 15:269–280PubMedCrossRefGoogle Scholar
  33. 33.
    Pommer AJ, Kuhlmann UC, Cooper A, Hemmings AM, Moore GR, James R, Kleanthous C (1999) J Biol Chem 274:27153–27160PubMedCrossRefGoogle Scholar
  34. 34.
    Hannan JP, Whittaker SBM, Hemmings AM, James R, Kleanthous C, Moore GR (2000) J Inorg Biochem 79:365–370PubMedCrossRefGoogle Scholar
  35. 35.
    Keeble AH, Hemmings AM, James R, Moore GR, Kleanthous C (2002) Biochemistry 41:10234–10244PubMedCrossRefGoogle Scholar
  36. 36.
    van den Bremer ETJ, Jiskoot W, James R, Moore GR, Kleanthous C, Heck AJR, Maier CS (2002) Protein Sci 11:1738–1752PubMedCrossRefGoogle Scholar
  37. 37.
    Hannan JP, Whittaker SB, Davy SL, Kuhlmann UC, Pommer AJ, Hemmings AM, James R, Kleanthous C, Moore GR (1999) Protein Sci 8:1711–1713PubMedCrossRefGoogle Scholar
  38. 38.
    van den Bremer ETJ, Keeble AH, Visser AJWG, van Hoek A, Kleanthous C, Heck AJR, Jiskoot W (2004) Biochemistry 43:4347–4355PubMedCrossRefGoogle Scholar
  39. 39.
    Ku W-Y, Liu Y-W, Hsu Y-C, Liao C-C, Liang P-H, Yuan HS, Chak K-F (2002) Nucleic Acids Res 30:1670–1678PubMedCrossRefGoogle Scholar
  40. 40.
    Shi Z, Chak K-F, Yuan HS (2005) J Biol Chem 280:24663–24668PubMedCrossRefGoogle Scholar
  41. 41.
    Li C-L, Hor L-I, Chang Z-F, Tsai L-C, Yang W-Z, Yuan HS (2003) EMBO J 22:4014–4025PubMedCrossRefGoogle Scholar
  42. 42.
    Gyurcsik B, Czene A (2011) Future Med Chem 3:1935–1966PubMedCrossRefGoogle Scholar
  43. 43.
    Tóth E, Czene A, Gyurcsik B, Otten H, Poulsen J-CN, Larsen S, Christensen HEM, Nagata K (2013) Acta Crystallogr Sect DGoogle Scholar
  44. 44.
    Limao-Vieira P, Giuliani A, Delwiche J, Parafita R, Mota R, Duflot D, Flament JP, Drage E, Cahillane P, Mason NJ, Hoffmann SV, Hubin-Franskin MJ (2006) Chem Phys 324:339–349CrossRefGoogle Scholar
  45. 45.
    Fahrni CJ, O’Halloran TV (1999) J Am Chem Soc 121:11448–11458CrossRefGoogle Scholar
  46. 46.
    Berendsen HJC, van der Spoel D, van Drunen R (1995) Comput Phys Commun 91:43–56CrossRefGoogle Scholar
  47. 47.
    Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) J Chem Theory Comput 4:435–447CrossRefGoogle Scholar
  48. 48.
    Oostenbrink C, Villa A, Mark AE, Van Gunsteren WF (2004) J Comput Chem 25(13):1656–1676PubMedCrossRefGoogle Scholar
  49. 49.
    Olsson MHM, Søndergaard CR, Rotkowski M, Jensen JH (2011) J Chem Theory Comput 7:525–537CrossRefGoogle Scholar
  50. 50.
    Stewart JJP (2008) MOPAC2009. Stewart Computational Chemistry, Colorado Springs. http://openmopac.net
  51. 51.
    Stewart JJP (2007) J Mol Model 13:1173–1213PubMedCrossRefGoogle Scholar
  52. 52.
    Stewart JJP (2009) J Mol Model 15:765–805Google Scholar
  53. 53.
    Stewart JJP (1996) Int J Quantum Chem 58:133–146CrossRefGoogle Scholar
  54. 54.
    Klamt A, Schüümann G (1993) J Chem Soc Perkin Trans 2 799–805Google Scholar
  55. 55.
    Anthony LC, Suzuki H, Filutowicz M (2004) J Microbiol Methods 58:243–250PubMedCrossRefGoogle Scholar
  56. 56.
    Levin KB, Dym O, Albeck S, Magdassi S, Keeble AH, Kleanthous C, Tawfik DS (2009) Nat Struct Mol Biol 16:1049–1055PubMedCrossRefGoogle Scholar
  57. 57.
    Wang Y-T (2009) Wright JD, Doudeva LG, Jhang H-C, Lim C, Yuan HS. J Am Chem Soc 131:17345–17353PubMedCrossRefGoogle Scholar
  58. 58.
    DeLano WL (2006) PyMOL version 0.99rc6. DeLano Scientific, San CarlosGoogle Scholar
  59. 59.
    Jecklin MC, Schauer S, Dumelin CE, Zenobi R (2009) J Mol Recognit 22:319–329PubMedCrossRefGoogle Scholar
  60. 60.
    Miles AJ, Wallace BA (2006) Chem Soc Rev 35:39–51PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2013

Authors and Affiliations

  • Anikó Czene
    • 1
    • 2
  • Eszter Németh
    • 1
    • 3
  • István G. Zóka
    • 1
  • Noémi I. Jakab-Simon
    • 1
    • 4
  • Tamás Körtvélyesi
    • 3
  • Kyosuke Nagata
    • 5
  • Hans E. M. Christensen
    • 4
  • Béla Gyurcsik
    • 1
    • 2
  1. 1.Department of Inorganic and Analytical ChemistryUniversity of SzegedSzegedHungary
  2. 2.Bioinorganic Chemistry Research Group of Hungarian Academy of SciencesSzegedHungary
  3. 3.Department of Physical Chemistry and Material SciencesUniversity of SzegedSzegedHungary
  4. 4.Department of ChemistryTechnical University of DenmarkKongens LyngbyDenmark
  5. 5.Department of Infection Biology, Graduate School of Comprehensive Human Sciences and Faculty of MedicineUniversity of TsukubaTsukubaJapan

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