The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154, and D158 of FlhA as most critical. These lie in a small, well conserved cytoplasmic domain of FlhA, located between trans-membrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.

Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the Nterminal peptide of the FliM motor protein (FliM N ). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY•FliM N with and without D13K-Y106W have shown FliM N bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliM N and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliM N interface; namely the closure of the 4-4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY•FliM N complex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliM N affinity. Activation entrained CheY fold stabilization to FliM N affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliM N interface according to phenotype. FliM N increased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far. Statement of SignificanceCheY affinity for FliM N , its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY•FliM N crystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliM N affinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliM N interface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliM N -tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

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 invivo 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 SignificanceThe chemotaxis phospho-protein CheY is activated at the flagellar motor by the Nterminus (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.