Dataset Information

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

ABSTRACT: We detail simulations and solution measurements to better understand the differences between the native and D13K-Y106W CheY crystal structures. We resolved the complex conformational landscapes by MD simulations with mutual information measures to determine the coupling between protein fragments. Protection experiments with XFMS (X-ray foot-printing with mass spectroscopy), a technique that probed sidechain solvent accessibility in contrast to deuterium exchange of backbone hydrogen atoms, supported the FliMN requirement for D13K-Y106W CheY activation reported by the crystal structures, and the MD allosteric network model. XFMS has a more straight-forward physical rationale than fluorescence quenching for reporting sidechain motions over time-resolved windows and is not limited by the size of the protein assembly. Further analysis of the MD trajectories resolved multiple CheY Y106 rotamer states. Inward orientation was temporally coupled to stabilization of both the CheY fold and the FliMN interface in the CheY·FliMN complex, but not in CheY alone. The coupling increased in D13K-Y106WCheY·FliMN. The formation of a distinct module that orchestrates CheY dynamicsto stabilize new surface topologies for possible second-stage binding to FliN was the signature of the fully activated D13K-Y106W CheY·FliMN state.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Escherichia Coli Bl21(de3)

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD020434 | Pride | 2020-09-21

REPOSITORIES: Pride

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

Wheatley Paige P Gupta Sayan S Pandini Alessandro A Chen Yan Y Petzold Christopher J CJ Ralston Corie Y CY Blair David F DF Khan Shahid S

Biophysical journal 20200815 6

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 (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliMN with and without D13K-Y106W have shown FliMN-bound CheY contains features of both active and inactive states. We used molecu ...[more]

PMID: 32891187

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