Proposed mechanisms of CheY allostery and flagellar motor switching

CheY is a member of the response regulator superfamily that controls the chemotactic swimming response of motile bacteria. Phosphorylation of Escherichia coli CheY increases its affinity for its target region at the FliM N-terminus, FliMN, by 20-fold. The association between phosphorylated CheY (CheY∼P) and FliM enables CheY∼P to modulate the rotational sense of the flagellar motor, a turbine-like molecular machine. The structure of the CheY∼P-FliMN complex had been solved by others, using BeF3- as a proxy for the phosphoryl moiety. However, the conformations of unphosphorylated and activated mutant versions of CheY in complex with FliMN remained unclear. We solved the crystallographic structures of unphosphorylated CheY and a constitutively active CheY mutant (CheYD13KY106W, or CheY**) in complex with FliMN. While binding target alone is sufficient to switch CheY** to the active conformation, it was not sufficient in unphosphorylated CheY. Instead, unphosphorylated CheY in complex with FliMN is found in a conformation intermediate to the active and inactive conformations. These results cast doubt on the longstanding assumption that CheY functions as a two-state switch. We next investigated the interaction of BeF3--activated CheY and a more complete fragment of FliM, derived from the hyperthermophillic bacterium Thermotoga maritima. This fragment, containing the first 229 residues of T. maritima FliM, has both the FliMN region and the FliM middle domain (FliMM). CheY was found to bind both regions of this FliM construct in solution. While the interaction with FliM N was anticipated, the CheY-FliMM interaction has not been reported previously. The CheY active site participates in this interface, perhaps allowing a catalytically active relative of FliM, FliY, to accelerate CheY dephosphorylation. FliG was also found to associate with FliMM using interfaces in both its C-terminal (FliGC) and middle domains (FliGM). FliGC and CheY bind with mutual exclusivity to a common surface centered on alpha-helix 2 of FliMM. FliG M binds to a different FliMM surface, and is unaffected by CheY binding. FliGC is known to generate torque by interacting directly with the Mot complexes. Therefore, these results suggest that CheY promotes flagellar motor reversal by displacing FliGC from FliM, which modulates the FliGC-MotA interaction.