| Shrnutí: | <p>Bacteria control their swimming direction by signalling from chemoreceptors via a small protein, CheY, to the rotary flagellar motor. <em>Rhodobacter sphaeroides</em> has a complex chemosensory network with two pathways, including three different CheYs controlling a stop-start motor. Deletions of these <i>che</i>Ys result in a non-chemotactic phenotype. CheY<sub>6</sub> is essential for chemotaxis, whereas CheY<sub>3</sub> and CheY<sub>4</sub> have some functional redundancies. Although CheY<sub>6</sub> alone can stop the motor, the presence of either CheY<sub>3</sub> or CheY<sub>4</sub> is required for a chemotactic phenotype. To date, little is known about how these three CheY proteins interact with the flagellar motor, or the switch mechanism used between the inactive and active states.</p> <p>Structural studies of CheY<sub>6</sub> using NMR experiments, highlighted a flexible loop region (residues S109-K118) that is not present in CheY<sub>3</sub>, CheY<sub>4</sub> or CheYs in other bacterial species. This elongated loop region was deleted (CheY<sub>6</sub>-ΔLoop), and <em>in vivo</em> studies were used to investigate its function. CheY<sub>6</sub> ΔLoop is folded, retains the ability to be phosphorylated by CheA<sub>3</sub>, localises at the cytoplasmic chemoreceptor cluster, but appears unable to stop the flagellar motor. Circular dichroism and NMR data suggest that CheY<sub>6</sub>-ΔLoop is a folded protein that shows similar peak shifts to wild type CheY<sub>6</sub> upon activation. In wild type CheY<sub>6</sub>, residues in this loop show chemical shift changes upon addition of the phosphoryl mimic BeF<sub>3</sub><sup>-</sup>, suggesting that the loop is involved in the activation mechanism. </p> <p>The switch mechanism of CheY<sub>6</sub> was probed using multidimensional NMR studies. The active state was mimicked using BeF<sub>3</sub><sup>-</sup> . Wild type CheY<sub>6</sub> was shown to undergo structural changes upon addition of BeF<sub>3</sub><sup>-</sup>. In particular, the β4 α4-loop and residues located near the phosphorylatable D56 show large changes in chemical shift. Superposition of this loop region revealed possible steric clashes with the N-terminus of FliM. S83 shows evidence of involvement in the switch mechanism. Residual dipolar coupling experiments suggest that the published crystal of CheY<sub>6</sub> in complex with CheA<sub>3</sub>, is more like the inactive conformation in solution. CheY has been shown to interact with the motor switch protein FliM in other bacterial species. CheY<sub>6</sub>-FliM interactions were probed using a combination of <em>in vitro</em> and <em>in vivo</em> studies. Bacterial two hybrid assays and NMR studies suggest that CheY<sub>6</sub> cannot interact with monomeric FliM. Single molecule total internal reflection microscopy revealed CheY<sub>6</sub> does interact with the motor <em>in vivo</em>.</p> <p>The data in combination suggests a model in which CheY<sub>6</sub>-P only interacts with FliM when it is part of the switch ring, and structural changes involved in stopping the motor depend on the large conformational changes in the β4-α4-loop.</p>
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