Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis

Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10902-7. doi: 10.1073/pnas.1001656107. Epub 2010 May 24.

Abstract

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 A) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Amino Acid Sequence
  • Bacteriological Techniques
  • Carbon Isotopes
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Histidine Kinase
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Multiprotein Complexes / chemistry
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Kinases / chemistry*
  • Protein Kinases / genetics
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics

Substances

  • Carbon Isotopes
  • Escherichia coli Proteins
  • Membrane Proteins
  • Multiprotein Complexes
  • Nitrogen Isotopes
  • QseC protein, E coli
  • Recombinant Proteins
  • KdpD protein, E coli
  • Protein Kinases
  • Histidine Kinase
  • arcB protein, E coli

Associated data

  • PDB/2KSD
  • PDB/2KSE
  • PDB/2KSF