Phosphorylation triggers domain separation in the DNA binding response regulator NarL.

DNA binding proteins of two-component signal transduction systems in microorganisms are activated by phosphorylation through an unknown mechanism. NarL is an example from the nitrate/nitrite signal transduction system of Escherichia coli. NarL consists of N- and C-terminal domains, the latter of which contains the DNA binding elements. To explore the mechanism of activation, single nitroxide side chains were introduced, one at a time, at nine different sites throughout the C-terminal domain to monitor the tertiary structure and the status of the surface in contact with the N-terminal domain. In addition, three pairs of doubly labeled proteins were prepared to monitor the interdomain distance using the magnetic dipolar interaction. The results of these site-directed spin-labeling studies reveal that phosphorylation at a distant site in the N-terminal domain triggers domain separation, likely by a hinge-bending motion. This in turn presents key elements of the C-terminal domain for docking to the DNA target in the configuration described in the recent crystal structure. The data also imply that a single conformation of unphosphorylated NarL exists in solution, and there is no detectable equilibrium between the closed and open conformations.