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A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli.

ABSTRACT: The motor that drives the flagellar filament of Escherichia coli contains three "switch" proteins (FliG, FliM, and FliN) that together determine the direction of rotation. Each is required, in addition, for flagellar assembly and for torque generation. These proteins interact in the Saccharomyces cerevisiae two-hybrid system: FliG interacts with FliM, FliM interacts with itself, and FliM interacts with FliN. The interaction between FliG and FliM has been subjected to mutational analysis. FliG (fused to the GAL4 DNA-binding domain) and FliM (fused to a GAL4 transcription activation domain) together activate transcription of a GAL4-dependent lacZ reporter gene. DNA encoding FliG was mutagenized by error-prone amplification with Taq polymerase, mutant fliG genes were cloned (as DNA-binding domain-fliG gene fusions) in S. cerevisiae by gap repair of plasmid DNA, and mutants exhibiting an interaction defect were isolated in a two-hybrid screen. The mutations were each mapped to the first, second, or last third of the fliG gene by multifragment cloning in vivo and then identified by DNA sequencing. In this way, we identified 18 interaction-defective and 15 silent (non-interaction-defective) fliG mutations. Several residues within the middle third of FliG are strongly involved in the FliG-FliM interaction, while residues near the N or C terminus are less important. This clustering, when compared with results of previous studies, suggests that the FliG-FliM interaction plays a central role in switching.

SUBMITTER: Marykwas DL

PROVIDER: S-EPMC177801 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli.

Marykwas D L DL Berg H C HC

Journal of bacteriology 19960301 5

The motor that drives the flagellar filament of Escherichia coli contains three "switch" proteins (FliG, FliM, and FliN) that together determine the direction of rotation. Each is required, in addition, for flagellar assembly and for torque generation. These proteins interact in the Saccharomyces cerevisiae two-hybrid system: FliG interacts with FliM, FliM interacts with itself, and FliM interacts with FliN. The interaction between FliG and FliM has been subjected to mutational analysis. FliG (f ...[more]

PMID: 8631704

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Project description: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 molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliMN 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 ⋅ FliMN 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 FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN 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.

Dataset Information

A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli.

Publications

A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli.

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