Advertisement

Biomolecular NMR Assignments

, Volume 9, Issue 2, pp 435–440 | Cite as

NMR assignment of intrinsically disordered self-processing module of the FrpC protein of Neisseria meningitidis

  • Vojtěch Kubáň
  • Jiří Nováček
  • Ladislav Bumba
  • Lukáš ŽídekEmail author
Article

Abstract

The self-processing module (SPM) is an internal segment of the FrpC protein (P415–F591) secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis during meningococcal infection of human upper respiratory tract. SPM mediates ‘protein trans-splicing’, a unique natural mechanism for editing of proteins, which involves a calcium-dependent autocatalytic cleavage of the peptide bond between D414 and P415 and covalent linkage of the cleaved fragment through its carboxy-terminal group of D414 to \(\epsilon\)-amino group of lysine residue within a neighboring polypeptide chain. We present an NMR resonance assignment of the calcium-free SPM, which displays characteristic features of intrinsically disordered proteins. Non-uniformly sampled 5D HN(CA)CONH, 4D HCBCACON, and HCBCANCO spectra were recorded to resolve poorly dispersed resonance frequencies of the disordered protein and 91 % of SPM residues were unambiguously assigned. Analysis of the chemical shifts revealed that two regions of the intrinsically disordered SPM (A95–S101 and R120–I127) have a tendency to form a helical structure, whereas the residues P1–D7 and G36–A40 have the propensity to adopt a \(\beta\)-structure.

Keywords

FrpC Self-processing module Neisseria meningitidis Intrinsically disordered proteins Sparse sampling Resolution-enhanced spectroscopy Resonance assignment 

Notes

Acknowledgments

The authors thank Iva Maršíková for technical help with protein purifications. This work was supported by the project GAP207-11-0717 of the Grant Agency of the Czech Republic. Additional thanks go to CEITEC—the Central European Institute of Technology with research infrastructure supported by the Project CZ.1.05/1.1.00/02.0068 financed from the European Regional Development Fund.

Ethical standard

The authors declare that the experiments comply with the current laws of the Czech Republic.

Conflicts of interest

The authors declare that they have no conflict of interest.

References

  1. Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed 44(20):3089–3092. doi: 10.1002/anie.200461794 CrossRefGoogle Scholar
  2. Bermel W, Bertini I, Felli IC, Gonnelli L, Koźmiński W, Piai A, Pierattelli R, Stanek J (2012) Speeding up sequence specific assignment of IDPs. J Biomol NMR 53(4):293–301. doi: 10.1007/s10858-012-9639-0 CrossRefGoogle Scholar
  3. Booy R, Kroll JS (1998) Bacterial meningitis and meningococcal infection. Curr Opin Pediatr 10(1):13–18CrossRefGoogle Scholar
  4. Bumba L, Sviridova E, Kutá Smatanová I, Řezáčová P, Veverka V (2014) Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis. Biomol NMR Assign 8(1):53–55. doi: 10.1007/s12104-012-9451-5 CrossRefGoogle Scholar
  5. Davies RL, Baillie S (2003) Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA. Vet Microbiol 92(3):263–279. doi: 10.1016/S0378-1135(02)00408-X CrossRefGoogle Scholar
  6. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293CrossRefGoogle Scholar
  7. Fullner KJ, Mekalanos JJ (2000) In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin. EMBO J 19(20):5315–5323. doi: 10.1093/emboj/19.20.5315 CrossRefGoogle Scholar
  8. Grifantini R, Sebastian S, Frigimelica E, Draghi M, Bartolini E, Muzzi A, Rappuoli R, Grandi G, Genco CA (2003) Identification of iron-activated and -repressed fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. Proc Natl Acad Sci USA 100(16):9542–9547. doi: 10.1073/pnas.1033001100 CrossRefADSGoogle Scholar
  9. Guibourdenche M, Høiby EA, Riou JY, Varaine F, Joguet C, Caugant DA (1996) Epidemics of serogroup a Neisseria meningitidis of subgroup III in Africa, 1989–94. Epidemiol Infect 116(2):115–120CrossRefGoogle Scholar
  10. Haesebrouck F, Chiers K, Van Overbeke I, Ducatelle R (1997) Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection. Vet Microbiol 58(2–4):239–249. doi: 10.1016/S0378-1135(97)00162-4 CrossRefGoogle Scholar
  11. Hart CA, Rogers TRF (1993) Meningococcal disease:—A review based on a symposium held on 11 July 1992 at the Liverpool School of Tropical Medicine. J Med Microbiol 39(1):3–25. doi: 10.1099/00222615-39-1-3 CrossRefGoogle Scholar
  12. Kazimierczuk K, Zawadzka A, Kozminski W, Zhukov I (2006) Random sampling of evolution time space and Fourier transform processing. J Biomol NMR 36(3):157–168. doi: 10.1007/s10858-006-9077-y CrossRefGoogle Scholar
  13. Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I (2007) Lineshapes and artifacts in Multidimensional Fourier Transform of arbitrary sampled NMR data sets. J Magn Reson 188(2):344–356. doi: 10.1016/j.jmr.2007.08.005 CrossRefADSGoogle Scholar
  14. Kazimierczuk K, Zawadzka A, Koźmiński W (2009) Narrow peaks and high dimensionalities: exploiting the advantages of random sampling. J Magn Reson 197(2):219–228. doi: 10.1016/j.jmr.2009.01.003 CrossRefADSGoogle Scholar
  15. Kazimierczuk K, Zawadzka-Kazimierczuk A, Koźmiński W (2010) Non-uniform frequency domain for optimal exploitation of non-uniform sampling. J Magn Reson 205(2):286–292. doi: 10.1016/j.jmr.2010.05.012 CrossRefADSGoogle Scholar
  16. Kjaergaard M, Poulsen FM (2011) Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution. J Biomol NMR 50(2):157–165. doi: 10.1007/s10858-011-9508-2 CrossRefGoogle Scholar
  17. Kjaergaard M, Sr Brander, Poulsen FM (2011) Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH. J Biomol NMR 49(2):139–149. doi: 10.1007/s10858-011-9472-x CrossRefGoogle Scholar
  18. Ladant D, Ullmann A (1999) Bordetella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 7(4):172–176. doi: 10.1016/S0966-842X(99)01468-7
  19. Lin W, Fullner KJ, Clayton R, Sexton JA, Rogers MB, Calia KE, Calderwood SB, Fraser C, Mekalanos JJ (1999) Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci USA 96(3):1071–1076. doi: 10.1073/pnas.96.3.1071 CrossRefADSGoogle Scholar
  20. Linhartová I, Bumba L, Mašín J, Basler M, Osička R, Kamanová J, Procházková K, Adkins I, Hejnová-Holubová J, Sadílková L, Morová J, Šebo P (2010) RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol Rev 34(6):1076–1112. doi: 10.1111/j.1574-6976.2010.00231.x CrossRefGoogle Scholar
  21. Marsh JA, Singh VK, Jia Z, Forman-Kay JD (2006) Sensitivity of secondary structure propensities to sequence differences between \(\alpha\)- and \(\gamma\)-synuclein: implications for fibrillation. Protein Sci 15(12):2795–2804. doi: 10.1110/ps.062465306 CrossRefGoogle Scholar
  22. Nováček J, Haba NY, Chill JH, Žídek L, Sklenář V (2012) 4D non-uniformly sampled HCBCACON and \(^1J(\text{ NC }^{\alpha })\)-selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins. J Biomol NMR 53(2):139–148. doi: 10.1007/s10858-012-9631-8 CrossRefGoogle Scholar
  23. Osička R, Kalmusová J, Křížová P, Šebo P (2001) Neisseria meningitidis RTX protein FrpC induces high levels of serum antibodies during invasive disease: Polymorphism of frpC alleles and purification of recombinant FrpC. Infect Immun 69(9):5509–5519. doi: 10.1128/IAI.69.9.5509-5519.2001 CrossRefGoogle Scholar
  24. Osička R, Procházková K, Šulc M, Linhartová I, Havlíček V, Šebo P (2004) A novel “Clip-and-link” activity of repeat in toxin (RTX) proteins from Gram-negative Pathogens: covalent protein cross-linking by an Asp-Lys isopeptide bond upon calcium-dependent processing at an Asp–Pro bond. J Biol Chem 279(24):24,944–24,956. doi: 10.1074/jbc.M314013200 CrossRefGoogle Scholar
  25. Pollard AJ, Faust SN, Levin M (1998) Meningitis and meningococcal septicaemia. J R Coll Physicians Lond 32(4):319–328Google Scholar
  26. Prochazkova K, Osicka R, Linhartova I, Halada P, Sulc M, Sebo P (2005) The Neisseria meningitidis outer membrane lipoprotein FrpD binds the RTX protein FrpC. J Biol Chem 280(5):3251–3258. doi: 10.1074/jbc.M411232200 CrossRefGoogle Scholar
  27. Sadilkova L, Osicka R, Sulc M, Linhartova I, Novak P, Sebo P (2008) Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC. Protein Sci 17(10):1834–1843. doi: 10.1110/ps.035733.108 CrossRefGoogle Scholar
  28. Thompson SA, Sparling PF (1993) The RTX cytotoxin-related FrpA protein of Neisseria meningitidis is secreted extracellularly by meningococci and by HlyBD+ Escherichia coli. Infect Immun 61(7):2906–2911Google Scholar
  29. Thompson SA, Wang LL, Sparling PF (1993) Cloning and nucleotide sequence of FrpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins. Mol Microbiol 9(1):85–96. doi: 10.1111/j.1365-2958.1993.tb01671.x CrossRefGoogle Scholar
  30. Thompson SA, Wang LL, West A, Sparling PF (1993b) Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins. J Bacteriol 175(3):811–818Google Scholar
  31. Welch RA (1991) Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol 5(3):521–528. doi: 10.1111/j.1365-2958.1991.tb00723.x CrossRefGoogle Scholar
  32. Welch RA (2001) RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology. Curr Top Microbiol Immunol 257:85–111Google Scholar
  33. Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfield E, Markley JL, Sykes BD (1995) \({}^1{\rm H }, {}^{13}{\rm C }\) and \({}^{15}{\rm N }\) chemical shift referencing in biomolecular NMR. J Biomol NMR 6(2):135–140. doi: 10.1007/BF00211777 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Vojtěch Kubáň
    • 1
  • Jiří Nováček
    • 1
  • Ladislav Bumba
    • 2
  • Lukáš Žídek
    • 1
    Email author
  1. 1.CEITECMasaryk UniversityBrnoCzech Republic
  2. 2.Institute of Microbiology of the ASCR, v. v. iPrague 4Czech Republic

Personalised recommendations