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Allosteric priming of E. coli CheY by the flagellar motor protein FliM

Phosphorylation of Escherichia coli CheY couple’s chemoreceptor output to flagellar motor response. The N-termini of FliM subunits (FliMN) in the flagellar rotor prime CheY to bind FliN thereby inducing cooperative switching, an essential feature of bacterial chemotaxis. We analyzed molecular dynamics {MD} trajectories to identify networks of residues involved in the long-range allosteric activation of CheY by FliMN. The CheY backbone was partitioned into four dynamically coordinated sectors, with activation tracked by changes in sector size and interactions. Bound FliMN closed the central α4-β4 loop hinge to strengthen correlations between sectors around its binding interface and D57 phosphorylation site. Inward W58 sidechain movements adjacent to the CheY D57 phosphorylation site were coupled to corresponding K91 and Y106 sidechain movements at the FliMN interface. Studies of the constitutively active CheY D13K-Y106W double mutant have related its structural changes with in-vivo signaling properties. The MD revealed that D13K-Y106W fused the phosphorylation site and FliMN binding sectors into a new surface-exposed sector and locked the 106W sidechain in the innermost rotamer configuration in CheY-FliMN complexes. X-ray foot-printing with mass spectroscopy exploited FliMN-CheY fusion proteins to validate the concerted sidechain internalization of W58, K91 and Y106 triggered by bound FliMN and increased by D13K-Y106W. Oxidation rate was correlated with the solvent accessible surface area, with K109, another central element of the allosteric relay, an outlier likely due to hydrogen bonding. The measurements indicated the fusion proteins were an effective mimic of the crystallized complexes used for the MD simulations. In absence of the D13K-Y106W mutations, CheY Y106 sampled multiple inward rotamer states, but their coupling to backbone dynamics required bound FliMN to prolongation inward state lifetimes by bound FliMN. Thus, as simulations have found for kinases, control of CheY activation by aromatic residue reorientation is more subtle than a binary ON-OFF switch. Statement of Significance The chemotaxis phospho-protein CheY is activated at the flagellar motor by the N-terminus (FliMN) of FliM subunits. Crystal structures of FliMN.CheY complexes with and without the phosphomimic D13K-Y106W double-mutation both have the residue 106 sidechain in the same IN orientation at the FliMN interface. Additional factors that explain activation were identified by atomistic simulations based on the crystal structures. Free CheY samples both IN and OUT Y106 rotamer states, but bound FliMN increases multiple IN-state lifetimes to alter backbone dynamics. D13K-Y106W triggers further alterations to select the W106 state seen in the crystals and create novel binding surfaces. Observed changes in sidechain position along the allosteric relay reported by the crystals were predicted and validated by X-ray foot-printing with mass-spectroscopy.