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Biomolecular NMR Assignments

, Volume 6, Issue 1, pp 9–13 | Cite as

NMR resonance assignment of the autoimmunity protein SpaI from Bacillus subtilis ATCC 6633

  • Nina Alexandra Christ
  • Elke Duchardt-Ferner
  • Stefanie Düsterhus
  • Peter Kötter
  • Karl-Dieter Entian
  • Jens WöhnertEmail author
Article

Abstract

Bacillus subtilis ATCC 6633 produces the lipid II targeting lantibiotic subtilin. For self-protection these gram-positive bacteria express a cluster of four self-immunity proteins named SpaIFEG. SpaI is a 16.8 kDa lipoprotein which is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Together with the ABC-transporter SpaFEG, SpaI protects the membrane from subtilin insertion and there is evidence for a direct interaction of SpaI with subtilin. As a prerequisite for further structural studies of SpaI and the SpaI/subtilin complex we report here the full 1H, 15N, 13C chemical shift assignment for a stable 14.9 kDa C-terminal fragment of SpaI.

Keywords

NMR-assignments Triple resonance experiments SpaI Self-immunity Lantibiotic Subtilin 

Notes

Acknowledgments

We are grateful to Dr. Christian Richter for help with the NMR experiments and to Sophie Bochmann for helpful discussions. We thank Prof. Volker Dötsch for the kind gift of the TEV-protease construct and Björn Meyer and Prof. Michael Karas for the MALDI-MS analysis of the C-terminal SpaI fragment. The eNMR project (European FP7 e-Infrastructure grant, contract no. 213010, www.enmr.eu), supported by the national GRID Initiatives of Italy, Germany and the Dutch BiG Grid project (Netherlands Organization for Scientific Research) is acknowledged for the use of web portals, computing and storage facilities. This project was supported by an Aventis Foundation professorship (to J.W.), the Center of Biomolecular Magnetic Resonance (BMRZ), the Cluster of Excellence “Macromolecular Complexes” and the Förderfonds of the Goethe University Frankfurt am Main.

References

  1. Bierbaum G, Sahl HG (2009) Lantibiotics: mode of action, biosynthesis and bioengineering. Curr Pharm Biotechnol 10:2–18CrossRefGoogle Scholar
  2. Breukink E, Wiedemann I, van Kraaij C, Kuipers OP, Sahl H, de Kruijff B (1999) Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286:2361–2364CrossRefGoogle Scholar
  3. Brötz H, Josten M, Wiedemann I, Schneider U, Götz F, Bierbaum G, Sahl HG (1998) Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol Microbiol 30:317–327CrossRefGoogle Scholar
  4. Eghbalnia HR, Wang L, Bahrami A, Assadi A, Markley JL (2005) Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements. J Biomol NMR 32:71–81CrossRefGoogle Scholar
  5. Engelke G, Gutowski-Eckel Z, Kiesau P, Siegers K, Hammelmann M, Entian KD (1994) Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3. Appl Environ Microbiol 60:814–825Google Scholar
  6. Gross E (1975) Subtilin and nisin: the chemistry and biology of peptides with alpha, beta-unsaturated amino acids, p. 31–42. In: R.Walter and J. Meienhofer (ed.), Peptides: chemistry, structure and biology: proceedings of the fourth American Peptide Symposium. Ann Arbor Sciecne, Ann Arbor, MI., pp. 31–42Google Scholar
  7. Halami PM, Stein T, Chandrashekar A, Entian K-D (2010) Maturation, processing of SpaI, the lipoprotein involved in subtilin immunity in Bacillus subtilis ATCC6633. Microbiol Res 165:183–189CrossRefGoogle Scholar
  8. Keller R (2004) The computer aided resonance tutorial. CANTINA Verlag, GoldauGoogle Scholar
  9. Klein C, Entian KD (1994) Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl Environ Microbiol 60:2793–2801Google Scholar
  10. Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wuthrich K (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. J Biomol NMR 12:1–23CrossRefGoogle Scholar
  11. Muchmore DC, McIntosh LP, Russell CB, Anderson de, Dahlquist FW (1989) Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol 177:44–73CrossRefGoogle Scholar
  12. Parisot J, Carey S, Breukink E, Chan WC, Narbad A, Bonev B (2008) Molecular mechanism of target recognition by Subtilin, a Class I Lanthionine Antibiotic. Antimicrob Agents Chemother 52:612–618CrossRefGoogle Scholar
  13. Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci USA 95:13585–13590ADSCrossRefGoogle Scholar
  14. Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158CrossRefGoogle Scholar
  15. Schnell N, Entian KD, Schneider U, Götz F, Zähner H, Kellner R, Jung G (1988) Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 333:276–278ADSCrossRefGoogle Scholar
  16. Schüller F, Benz R, Sahl HG (1989) The peptide antibiotic subtilin acts by formation of voltage-dependent multi-state pores in bacterial and artificial membranes. Eur J Biochem 182:181–186CrossRefGoogle Scholar
  17. Shen Y, Bax A (2010) Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts. J Biomol NMR 46:199–204CrossRefGoogle Scholar
  18. Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223CrossRefGoogle Scholar
  19. Stein T, Heinzmann S, Düsterhus S, Borchert S, Entian K-D (2005) Expression and functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive host Bacillus subtilis MO1099. J Bacteriol 187:822–828CrossRefGoogle Scholar
  20. Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1:749–754CrossRefGoogle Scholar
  21. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696CrossRefGoogle Scholar
  22. Willey JM, van der Donk WA (2007) Lantibiotics: peptides of diverse structure and function. Annu Rev Microbiol 61:477–501CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Nina Alexandra Christ
    • 1
    • 2
  • Elke Duchardt-Ferner
    • 1
    • 2
  • Stefanie Düsterhus
    • 1
  • Peter Kötter
    • 1
  • Karl-Dieter Entian
    • 1
    • 3
  • Jens Wöhnert
    • 1
    • 2
    • 3
    Email author
  1. 1.Institut für Molekulare BiowissenschaftenJohann-Wolfgang-Goethe-Universität Frankfurt/M.FrankfurtGermany
  2. 2.Center of Biomolecular Magnetic Resonance (BMRZ)Johann-Wolfgang-Goethe-Universität Frankfurt/M.FrankfurtGermany
  3. 3.Cluster of Excellence “Macromolecular Complexes”Johann-Wolfgang-Goethe-UniversitätFrankfurtGermany

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