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Comparative Study
. 2010 Jun 15;107(24):10902-7.
doi: 10.1073/pnas.1001656107. Epub 2010 May 24.

Membrane Domain Structures of Three Classes of Histidine Kinase Receptors by Cell-Free Expression and Rapid NMR Analysis

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Free PMC article
Comparative Study

Membrane Domain Structures of Three Classes of Histidine Kinase Receptors by Cell-Free Expression and Rapid NMR Analysis

Innokentiy Maslennikov et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Three classes of HKRs. (A) Schematic representation of TM domains of three classes of HKRs and (B) ribbon representation of 3D structures of the TM domains of E. coli HKRs ArcB, QseC, and KdpD.
Fig. 2.
Fig. 2.
Verification of fold of ArcB(1-115), produced by CF system. (A) Overlay of 13C DARR-NMR spectra (213.765 MHz) of uniformly 13C-labeled ArcB(1-115) expressed in p-CF reaction. Black contours correspond to the spectra of the washed precipitant of the p-CF reaction. Red contours correspond to the spectra of the ArcB(1-115) sample lyophilized after the precipitant was solubilized in 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) . The green and cyan lines correspond to random coil formula image, formula image chemical shifts for valine (Val) and alanine (Ala), respectively; arrows show regions of chemical shifts corresponding to the α-helical conformation. (B and C) [1H-15N]-TROSY-HSQC spectra of 15N-labeled ArcB(1-115) expressed (B) by p-CF synthesis and in (C) E coli. (B) The precipitated protein was washed and solubilized in 5% LMPG. (C) The protein was extracted and purified from cell membrane with the FC-12 detergent and the detergent was exchanged to LMPG. Cross-peaks denoted by red arrows correspond to the tag linker residues in the E. coli-expressed protein.
Fig. 3.
Fig. 3.
Comparison of performance of the CF system with the standard E. coli system. SDS-PAGE shows marker (M); CF reaction mixture (RM) before reaction at 0 h (1); CF RM after ArcB(1-115) expression at 15 h (2); precipitate after ArcB(1-115) p-CF (3); E. coli (EC) expressed ArcB(1-115) after extraction, purification, Tag cleavage, size-exclusion chromatography (SEC), detergent exchange on Q-sepharose, and concentration (4); arrow indicates ArcB(1-115).
Fig. 4.
Fig. 4.
The CDL strategy for the “point-directed assignment” of NMR spectra. (A) The [1H-15N] cross-peak in HSQC will appear only if the second residue in a pair is 15N labeled (second box tagged 1). The [1H-15N] cross-peak in the HSQC spectrum and the [1H-15N-13C] cross-peak in the HNCO spectrum will both appear only if the peptide group is double [13C-15N]-labeled (third box tagged 2). (B) Dual 15N/13C combinatorial selective labeling scheme designed specifically for backbone assignment of KdpD(397-502) (see details in SI Text). (C) Assignment of 1H-15N cross-peaks of KdpD(397-502) using a combinatorial scheme of selective 13C, 15N labeling, presented in panel B. The overlays of [1H-15N]-TROSY-HSQC (red contours) and [1H-15N] projection of TROSY-HNCO (blue contours) spectra are shown for each sample (I–VI). Absence of a cross-peak (tag “0”), a cross-peak present in TROSY only (tag “1”), and cross-peaks present in both the TROSY and the HNCO spectra (tag “2”) in each combinatorially labeled sample determine the code (sequence of the tags) for every cross-peak A, B, and C in a uniformly labeled sample. The scheme-designed code which is identical with the code determined from the recorded spectra defines a pair of amino acids for the formula image, formula image, and formula image resonances.
Fig. 5.
Fig. 5.
Twenty superimposed structures of the TM domains of (A) ArcB(1-115), (B) QseC(1-185), and (C) KdpD(397-502). Backbones are shown for the stable regions: ArcB(1-115), residues 20-83; QseC(1-185), residues 10-38; and 156-185 and KdpD(397-502), residues 397-502. Consecutive TM helices are colored in the following order: green, blue, orange, and coral. Structures on the right are rotated 90° vertically.

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