Solution structure of NPr, a bacterial signal-transducing protein that controls the phosphorylation state of the potassium transporter-regulating protein IIA^Ntr

A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EI^Ntr, NPr, and IIA^Ntr, was discovered recently to regulate the uptake of K⁺ in Escherichia coli. In particular, dephosphorylated IIA^Ntr inhibits the activity of the K⁺ transporter TrkA. Since the phosphorylation state of IIA ^Ntr is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shift-derived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three α-helices and four β-strands with the three helices all packed on the same face of the β-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the Nε2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.