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 × 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 IIA^Ntr 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 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 Nδ1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway. [ABSTRACT FROM AUTHOR]