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Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 103–106 | Cite as

1H, 15N, 13C backbone resonance assignment of the C-terminal domain of enzyme I from Thermoanaerobacter tengcongensis

  • Rochelle Rea Dotas
  • Vincenzo Venditti
Article
  • 83 Downloads

Abstract

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone 1H, 15N and 13C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the 1H–15N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.

Keywords

Enzyme I Thermophilic bacteria Backbone resonance assignment Bacterial phosphotransferase system Transverse relaxation optimized spectroscopy Conformational dynamics 

Notes

Acknowledgements

This work was supported by startup funding from Iowa State University (V.V.).

References

  1. Clore GM, Gronenborn AM (1998) Determining the structures of large proteins and protein complexes by NMR. Trends Biotechnol 16:22–34CrossRefGoogle Scholar
  2. Clore GM, Venditti V (2013) Structure, dynamics and biophysics of the cytoplasmic protein-protein complexes of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Trends Biochem Sci 38:515–530. doi: 10.1016/j.tibs.2013.08.003 CrossRefGoogle Scholar
  3. 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 6:277–293CrossRefGoogle Scholar
  4. Deutscher J, Ake FM, Derkaoui M, Zebre AC, Cao TN, Bouraoui H, Kentache T, Mokhtari A, Milohanic E, Joyet P (2014) The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions. Microbiol Mol Biol Rev 78:231–256. doi: 10.1128/MMBR.00001-14 CrossRefGoogle Scholar
  5. Navdaeva V, Zurbriggen A, Waltersperger S, Schneider P, Oberholzer AE, Bahler P, Bachler C, Grieder A, Baumann U, Erni B (2011) Phosphoenolpyruvate: sugar phosphotransferase system from the hyperthermophilic Thermoanaerobacter tengcongensis. BioChemistry 50:1184–1193CrossRefGoogle Scholar
  6. Oberholzer AE, Bumann M, Schneider P, Bachler C, Siebold C, Baumann U, Erni B (2005) Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS). J Mol Biol 346:521–532CrossRefGoogle Scholar
  7. Patel HV, Vyas KA, Savtchenko R, Roseman S (2006) The monomer/dimer transition of enzyme I of the Escherichia coli phosphotransferase system. J Biol Chem 281:17570–17578. doi: 10.1074/jbc.M508965200 CrossRefGoogle Scholar
  8. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar
  9. Postma PW, Lengeler JW, Jacobson GR (1996) Phosphoenolpyruvate:carbohydrate phosphotransferase systems. In: Neidhardt FC, Lin EC, Curtiss R (eds) Escherichia coli and Salmonella: cellular and molecular biology, pp 1149–1174. ASM Press, Washington, DCGoogle Scholar
  10. Schwieters CD, Suh JY, Grishaev A, Ghirlando R, Takayama Y, Clore GM (2010) Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering. J Am Chem Soc 132:13026–13045CrossRefGoogle Scholar
  11. 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
  12. Teplyakov A, Lim K, Zhu PP, Kapadia G, Chen CC, Schwartz J, Howard A, Reddy PT, Peterkofsky A, Herzberg O (2006) Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein. Proc Natl Acad Sci USA 103:16218–16223ADSCrossRefGoogle Scholar
  13. Tugarinov V, Muhandiram R, Ayed A, Kay LE (2002) Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. J Am Chem Soc 124:10025–10035CrossRefGoogle Scholar
  14. Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley J (2008) BioMagResBank. Nucleic Acids Res 36:D402–D408Google Scholar
  15. Venditti V, Clore GM (2012) Conformational selection and substrate binding regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli enzyme I. J Biol Chem 287:26989–26998. doi: 10.1074/jbc.M112.382291 CrossRefGoogle Scholar
  16. Venditti V, Ghirlando R, Clore GM (2013) Structural basis for enzyme I inhibition by alpha-ketoglutarate. ACS Chem Biol 8:1232–1240. doi: 10.1021/cb400027q CrossRefGoogle Scholar
  17. Venditti V, Schwieters CD, Grishaev A, Clore GM (2015a) Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering. Proc Natl Acad Sci USA 112:11565–11570. doi: 10.1073/pnas.1515366112 ADSCrossRefGoogle Scholar
  18. Venditti V, Tugarinov V, Schwieters CD, Grishaev A, Clore GM (2015b) Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial enzyme I. Nat Commun 6:5960. doi: 10.1038/ncomms6960 ADSCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of ChemistryIowa State UniversityAmesUSA
  2. 2.Roy J. Carver Department of Biochemistry, Biophysics and Molecular BiologyIowa State UniversityAmesUSA

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