Allosteric Priming of E. coli CheY by the Flagellar Motor Protein FliM.

ABSTRACT: Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal 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 D57 phosphorylation site and the FliM_N interface, namely the closure of the α4-β4 hinge and inward rotation of Y- or W106 with W58. We used hydroxy-radical foot printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other side chains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay side chains 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- or W106 side-chain 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 K13-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.

SUBMITTER: Wheatley P 

PROVIDER: S-EPMC7499101 | biostudies-literature |

REPOSITORIES: biostudies-literature