Journal of Biological Chemistry
Volume 295, Issue 50, 11 December 2020, Pages 16960-16974
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Signal Transduction
The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

https://doi.org/10.1074/jbc.RA120.014083Get rights and content
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The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in “cis-mediated” style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Flagellar synthesis
Vibrio cholera
bacterial enhancer binding protein
σ54-dependent transcription
oligomeric assembly
ATPase activity
mutagenesis
crystal structures
sequence analysis
bacterial transcription
ATPases associated with diverse cellular activities (AAA)
site-directed mutagenesis
oligomerization
cyclic di-GMP (c-di-GMP)

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This article contains supporting information.

Author contributions—S. C. and B. G. data curation; S. C. and S. A. formal analysis; S. C. and B. G. validation; S. C., M. B., S. D., S. A., and T. C. investigation; S. C., M. B., S. D., and T. C. methodology; B. G. software; J. D. conceptualization; J. D. supervision; J. D. funding acquisition; J. D. writing-original draft; J. D. project administration.

Funding and additional information—This work was supported by DBT Grant BT/03/IYBA/2010 of Govt. of India, DST Grant SR/FST/COLLEGE-014/2010(C), and WBDBT BOOST Grant 335/WBBDC/1P-2/2013 for infrastructural support.

Conflict of interest—The authors declare that they have no conflicts of interest related to the contents of this article.

Present address for Maitree Biswas: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Present address for Sanjay Dey: CNRS UMR 7104-Inserm U 1258, France.

Present address for Shubhangi Agarwal: Department of Microbiology, University of Hohenheim, Stuttgart, Germany.

Abbreviations—The abbreviations used are:

    bEBP

    bacterial enhancer binding protein

    AAA+

    ATPases associated with various cellular activities

    Nt

    nucleotide

    PDB

    Protein Data Bank

    AMP-PNP

    adenosine 5′-(β,γ-imino)triphosphate

    c-di-GMP

    cyclic di-guanosine monophosphate

    SEC

    size exclusion chromatography

    R

    regulator

    RMSD

    root mean square deviation.