PMID:12614149
| Citation |
Zhang, JH, Xiao, G, Gunsalus, RP and Hubbell, WL (2003) Phosphorylation triggers domain separation in the DNA binding response regulator NarL. Biochemistry 42:2552-9 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1021/bi0272205 |
| Keywords |
Amino Acid Substitution/genetics; Aspartic Acid/genetics; Crystallization; Cyclic N-Oxides/chemistry; DNA/chemistry; DNA/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Phosphorylation; Protein Binding/genetics; Protein Structure, Secondary/genetics; Protein Structure, Tertiary/genetics; Solutions; Spectrometry, Mass, Electrospray Ionization; Spin Labels; Surface Properties; Thermodynamics |
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