title: Phosphorylation triggers domain separation in the DNA binding response regulator NarL.
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.
authors.affiliation: Jules Stein Eye Institute, Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1489, USA.
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Zhang
authors.forename: Gaoping
authors.initials: G
authors.lastname: Xiao
authors.forename: Robert P
authors.initials: RP
authors.lastname: Gunsalus
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D003460:Crystallization; D003497:Cyclic N-Oxides / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D013113:Spin Labels; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D003497:Cyclic N-Oxides; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012996:Solutions; D013113:Spin Labels; C069780:NarL protein, E coli; D001224:Aspartic Acid; D004247:DNA