ATP-Induced Structural Remodeling in the Antiactivator FleN Enables Formation of the Functional Dimeric Form

Abstract

FleN, a P loop ATPase is vital for maintaining a monotrichous phenotype in Pseudomonas aeruginosa. FleN exhibits antagonistic activity against FleQ, the master transcriptional regulator of flagellar genes. Crystal structures of FleN in the apo form (1.66 Å) and in complex with β,γ-imidoadenosine 5'-triphosphate (1.55 Å) reveal that it undergoes drastic conformational changes on ATP binding to attain a structure capable of dimerization. Mutations of the residues that stabilize the binding of ATP were defective in their ability to dimerize and do not inhibit ATP hydrolysis by FleQ. Conversely, the catalytic mutant of FleN, was an efficient inhibitor. These observations posit that the dimer is the functional form of FleN and it is nucleotide binding and not hydrolysis by FleN that is necessary to exert an antagonistic effect against FleQ. Our study shows that ATP-induced dimerization may be a strategy to achieve reversible inhibition of FleQ to fine-tune the function of this activator to an optimal level.

The antiactivator FleN uses an allosteric mechanism to regulate σ 54 -dependent expression of flagellar genes in Pseudomonas aeruginosa

“…As FleN is a known inhibitor of the ATPase activity of FleQ ( 19 ), we further examined the ability of FleN to inhibit the ATPase activity of different domain constructs of FleQ. FleN reduces the ATPase activity of FleQ FL , FleQ AAA+ , and FleQ RECAAA+ by 80%, and the residual activity is similar to the ATPase activity of FleN ( Fig.…”

“…It has been shown that ATP-mediated dimerization in FleN is essential for its antagonist effect over FleQ. It exerts this effect without affecting the DNA binding ability of FleQ ( 18 , 19 ). However, the lack of structural data on the protein-protein complex impedes a detailed understanding of the mechanism of transcription inhibition by antiactivators of σ 54 regulators in general and by FleN in particular.…”

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression

“…FlhG is homologous to the MinD/ParA-type ATPases and shares significant structural and functional similarity (8,10); indeed, in C. jejuni FlhG plays the role of MinD in determining the site of cell division (11). Like MinD (12,13), FlhG forms ATPdependent homodimers that interact with the inner membrane through a C-terminal amphipathic helix or a membranetargeting sequence (MTS) (8,10). FlhG acts in concert with the signal recognition particle-GTPase FlhF and stimulates the GTPase activity of FlhF (14)(15)(16).…”

Partially Reciprocal Replacement of FlrA and FlrC in Regulation of Shewanella oneidensis Flagellar Biosynthesis

“…Studies of P. aeruginosa FleN, an ortholog of FlhG, have established that FleN inhibits FleQ (the FlrA ortholog) by direct interaction (32)(33)(34). For Shewanella spp., a similar mechanism for the antagonistic effect of FlhG against FlrA was proposed recently (35).…”

“…Importantly, we found that the multiflagellum phenotype resulting from the deletion of flhG or flhFG is due to enhanced activity of FlrA. The association of FlhG with FlrA was initially established with the P. aeruginosa FlhG ortholog, FleN; FlhG and FleN share 35% sequence identity and are highly similar in structure (32)(33)(34). FleN exerts an containing 20 mM imidazole, the His-tagged FlhF variants were eluted in elution buffer containing 100 mM imidazole.…”