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

¹ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA² Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA

(RECEIVED November 11, 2004; FINAL REVISION December 9, 2004; ACCEPTED December 9, 2004)

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 (`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 N1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway.

Keywords: NMR; translational diffusion coefficient; heteronuclear NOE; residual dipolar coupling; IIA^Ntr; NPr; Escherichia coli; structure-based functional discovery

Abbreviations: PTS, phosphoenolpyruvate:sugar phosphotransferase system • PEP, phosphoenolpyruvate • IIA^Glc, glucose-specific enzyme IIA • HPr, histidine-containing phosphocarrier protein • EI, enzyme I • EI^Ntr, nitrogen-related enzyme I • NPr, nitrogen-related HPr • IIA^Ntr, nitrogen-related enzyme IIA • IIA^Ntr(9–163), IIA^Ntr with N-terminal residues 1–8 deleted • NMR, nuclear magnetic resonance • IPAP, in-phase antiphase • HSQC, heteronuclear single quantum coherence • NOE, nuclear Overhauser effect • RDC, residual dipolar coupling • SVD, single value decomposition • PCR, polymerase chain reaction • IPTG, isopropyl--D-thiogalactopyranoside • PMSF, phenylmethylsulfonyl fluoride • SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041232805.

Reprint requests to: Guangshun Wang, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA; e-mail: gwang{at}unmc.edu; fax: (402) 559-4651.

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