A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase

Anna Koganitsky, Dmitry Tworowski, Tali Dadosh, Gary Cecchini, Michael Eisenbach

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliG—one of the motor's switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliGcw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdA–FliGcw interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA's conformation to one that can bind to FliGcw. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliGcw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG0 between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.

Original languageEnglish
Pages (from-to)3662-3676
Number of pages15
JournalJournal of Molecular Biology
Volume431
Issue number19
DOIs
StatePublished - 6 Sep 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2019 Elsevier Ltd

Funding

We thank Dr. Hillary Voet, a statistician, for the statistical advices, Dr. Shira Albec from the Hollander Center for Structural Proteomics for protein purification, Dr. Irina Shin for carrying out the MST measurements, and Vladimir Kiss for assistance in fluorescence microscopy. G.C. was supported by the National Institutes of Health award GM61606 . G.C. is also the recipient of a Senior Research Career Scientist award 1K6B004215 from the Department of Veterans Affairs . We thank Dr. Hillary Voet, a statistician, for the statistical advices, Dr. Shira Albec from the Hollander Center for Structural Proteomics for protein purification, Dr. Irina Shin for carrying out the MST measurements, and Vladimir Kiss for assistance in fluorescence microscopy. G.C. was supported by the National Institutes of Health award GM61606. G.C. is also the recipient of a Senior Research Career Scientist award 1K6B004215 from the Department of Veterans Affairs.

FundersFunder number
Hollander Center for Structural Proteomics
National Institutes of Health1K6B004215, GM61606
U.S. Department of Veterans AffairsIK6BX004215

    Keywords

    • FliG
    • anaerobic complex II
    • flagellar motor
    • protein electrostatics
    • respiratory complexes

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