Allosteric priming of E. coli CheY by the flagellar motor protein FliM
Authors listPaige Wheatley Sayan Gupta Alessandro Pandini Yan Chen Christopher J Petzold Corie Y Ralston David F Blair Shahid Khan
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). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliM with and without D13K-Y106W have shown FliM-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 and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliM 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 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 affinity. Activation entrained CheY fold stabilization to FliM affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliM interface according to phenotype. FliM 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.
Journal Biophysical Journal
Issue number 6