Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB from Escherichia coli

J Phys Chem B. 2018 Jun 7;122(22):5749-5757. doi: 10.1021/acs.jpcb.8b01034. Epub 2018 May 15.

Abstract

PhoB is a response regulator of PhoR/PhoB two-component system from Escherichia coli that is involved in the environmental phosphate regulation. It has been reported that the N-terminal receiver domain (PhoBN) forms a dimer using the α1-α5 face in the apo state and a dimer using the α4-β5-α5 face in the active state investigated by X-ray crystallography. However, it is not clear whether the conformational switch of the dimer is dependent on phosphorylation. Here, we report the NMR studies of PhoBN in solution in its apo form. We observed that the secondary structural fragments of apo PhoBN characterized by NMR are almost the same as those determined by crystallography, but the NMR spectrum of PhoBN shows inhomogeneous amide signals. Concentration dilution experiments and backbone relaxation parameters showed that PhoBN exists in equilibrium between monomer and dimer states. Using paramagnetic relaxation enhancement experiments, we demonstrated that the dimer of apo PhoBN forms several transient dimer interfaces in solution. This finding suggested that, in addition to the monomer-to-dimer exchange, the inactive conformation of PhoBN has different domain arrangements, which are independent of phosphorylation. It provides an experimental data for the conformational selection mechanism of the phosphorylation of PhoBN.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Dimerization
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphorylation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Escherichia coli Proteins
  • PhoB protein, E coli
  • Recombinant Proteins
  • Transcription Factors