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. 2013 Sep;89(5):831-41.
doi: 10.1111/mmi.12309. Epub 2013 Jul 30.

The Mobility of Two Kinase Domains in the Escherichia Coli Chemoreceptor Array Varies With Signalling State

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The Mobility of Two Kinase Domains in the Escherichia Coli Chemoreceptor Array Varies With Signalling State

Ariane Briegel et al. Mol Microbiol. .
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Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.


Fig. 1
Fig. 1. Structures of MCP receptors, CheA kinase, and ternary signaling complexes
(A) Input-output signaling in chemoreceptors of the MCP family. These molecules function as homodimers; the subunits are approximately 500 residues in length and mainly alpha-helical in secondary structure. The approximate positions of amino acid changes used to modify receptor output state in this work are shown. (B) Domain organization of CheA. This autokinase functions as a homodimer. Autophosphorylation is a trans reaction, involving interaction of the phosphorylation site domain in one subunit with the ATP-binding domain of the other (Wolfe & Stewart, 1993). Two CheA variants used in the present work are shown at the right. (C) The core signaling units of bacterial chemoreceptors. MCP molecules assemble into trimers of dimers through interactions between their highly conserved cytoplasmic tips (Kim et al., 1999, Studdert & Parkinson, 2004). Two trimers share and control one CheA dimer through binding interactions to its two P5 domains (one is hidden behind the trimer on the right) and to two P5-like CheW coupling proteins (one is hidden behind the trimer on the left). The CheW proteins each interact with a P5 domain, providing additional conformational control connections to the receptors. These core complexes assemble into higher order arrays through additional P5-CheW interactions (Briegel et al., 2012).
Fig. 2
Fig. 2. No large-scale differences are evident in mutant receptor arrays
Representative tomographic slices and corresponding power spectra of kinase-inactive (left column) and kinase-active receptor mutant arrays (right column). All arrays have comparable hexagonal organization with 12 nm spacing. Scale bar (bottom left panel): 30 nm.
Fig. 3
Fig. 3. Keel electron density varies in ternary signaling complexes in different output states
Image averages are shown for signaling complexes of eight Tsr variants with the amino acid changes listed across the top: wild-type receptor subunits (QEQE) have Q residues at two of the four methylation sites; the EEEE and QQQQ variants mimic the fully unmethylated or fully methylated forms of the receptor. The A413T, P221D, M222R, G235E and I241E mutant receptors are variants of the QEQE wild-type. Receptor crystal structures (purple) were fitted into sub-tomogram averages by MDFF for reference and alignment. An extra keel-like density, most prominent in the A413T complexes, is seen below the receptors in the side views (top row). The numbers below the keels give the volume of the keel density as a percentage of the total volume occupied by two trimers plus the keel at an appropriate threshold (shown in blue). Bottom row: top views showing keel densities connecting adjacent trimers (arrowheads), alternating with gaps around the hexagons (asterisks).
Fig. 4
Fig. 4. Keel electron density comprises CheA subunits P1 and P2
From left to right: side views, side views rotated 90°, top views. A-F: Signaling complexes of the A413T receptor that compare the keels seen with wild-type CheA (gray) to those seen with CheA mutants (blue). (A, B, C) wild-type CheA and CheAΔ(P1-P2) signaling complexes. (D, E, F) wild-type CheA and CheAΔP2 signaling complexes. Black arrows point to clamp-like structures on each side of the keel that probably contain the P1 domain and possibly part of the P4 domain with which it interacts. (G, H, I) Signaling complexes of the P221D receptor that compare the keels seen with wild-type CheA (light gray) to those seen with CheAΔ(P1-P2) (cyan).
Fig. 5
Fig. 5. CheA protease sensitivity corresponds to keel size
Western blots of mutant strains without (−) or with (+) treatment with proteinase K. Top: α-CheA. Arrows indicate long and short isoforms of CheA. Bottom: loading control (proteinase K-resistant background band (*) detected by α-β-lactamase). Values at bottom indicate percent of CheAL signal remaining after treatment for each strain.

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