NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr

Guangshun Wang, Alan Peterkofsky, Paul A. Keifer, Xia Li

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The solution form of IIANtr from Escherichia coli and its interaction with its partner protein, NPr, were characterized by nuclear magnetic resonance (NMR) spectroscopy. The diffusion coefficient of the protein (1.13 × 10-6 cm/sec) falls between that of HPr (∼ 9 kDa) and the N-terminal domain of E. coli enzyme I (∼ 30 kDa), indicating that the functional form of IIANtr is a monomer (∼ 18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data of IIA Ntr (covering residues 11-155) measured in the absence and presence of a 4% polyethyleneglycol-hexanol liquid crystal alignment medium fit well to the coordinates of both molecule A and molecule B of the dimeric crystal structure, indicating that the 3D structures in solution and in the crystal are indeed similar for that protein region. However, only molecule A possesses an N-terminal helix identical to that derived from chemical shifts of IIA Ntr in solution. Further, the 15N heteronuclear nuclear Overhauser effect (NOE) data also support molecule A as the representative structure in solution, with the terminal residues 1-8 and 158-163 more mobile. Chemical shift mapping identified the surface on IIANtr for NPr binding. Residues Gly61, Asp115, Ser125, Thr156, and nearby regions of IIA Ntr are more perturbed and participate in interaction with NPr. The active-site His73 of IIANtr for phosphoryl transfer was found in the Nδ1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway.

Original languageEnglish (US)
Pages (from-to)1082-1090
Number of pages9
JournalProtein Science
Volume14
Issue number4
DOIs
StatePublished - Apr 2005

Keywords

  • Escherichia coli
  • Heteronuclear NOE
  • IIA
  • NMR
  • NPr
  • Residual dipolar coupling
  • Structure-based functional discovery
  • Translational diffusion coefficient

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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