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

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2010-06, Vol.107 (24), p.10902-10907
Main Authors: Maslennikov, Innokentiy, Klammt, Christian, Hwang, Eunha, Kefala, Georgia, Okamura, Mizuki, Esquivies, Luis, Mörs, Karsten, Glaubitz, Clemens, Kwiatkowski, Witek, Jeon, Young Ho, Choe, Senyon
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Bacteriological Techniques
Biochemistry
Biological Sciences
Carbon Isotopes
Cells
Crystal structure
Data banks
Detergents
Escherichia coli
Escherichia coli - chemistry
Escherichia coli - genetics
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Histidine Kinase
Isotopic labeling
Kinases
Membrane proteins
Membrane Proteins - chemistry
Membrane Proteins - genetics
Membranes
Models, Molecular
Molecular Sequence Data
Multiprotein Complexes - chemistry
N.M.R
Nitrogen Isotopes
NMR
Nuclear magnetic resonance
Nuclear Magnetic Resonance, Biomolecular
Precipitates
Probes
Protein biosynthesis
Protein Kinases - chemistry
Protein Kinases - genetics
Protein Structure, Tertiary
Protein synthesis
Receptors
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Sensors
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Summary: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 Å) 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1001656107