The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and...

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 (FlrC^C) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrC^C 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 FlrC^C-Y290A and FlrC^C-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrC^C-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrC^C-Y290A. We postulated that FlrC^C 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 FlrC^C Excess cyclic-di-GMP repressed ATPase activity of FlrC^C 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.