NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr.
The solution form of IIA(Ntr) 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 x 10(-6) cm/sec) falls between that of HPr (approximately 9 kDa) and the N-terminal domain of E. coli enzyme I (approximately 30 kDa), indicating that the functional form of IIA(Ntr) is a monomer (approximately 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 (15)N 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 IIA(Ntr) 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 IIA(Ntr) for phosphoryl transfer was found in the Ndelta1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway.
Wang
Guangshun
G
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA. gwang@unmc.edu
Peterkofsky
Alan
A
Keifer
Paul A
PA
Li
Xia
X
D002352:Carrier Proteins / Q000737:chemistry*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D028044:Phosphate-Binding Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D017433:Protein Structure, Secondary
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C502371:NPr protein, E coli; D028044:Phosphate-Binding Proteins; C487562:IIA(Ntr) protein, E coli; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System
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