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

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. [Display omitted] •FleN, a P loop ATPase, inhibits ATP hydrolysis by the transcription regulator FleQ•ATP binding by FleN elicits conformational changes that enable formation of a dimer•Nucleotide binding is necessary and sufficient for antiactivator function of FleN•Reversible dimerization of FleN regulates FleQ and ensures monoflagellate status FleN is an antiactivator of FleQ, a global transcriptional regulator of flagellar and biofilm genes in Pseudomonas aeruginosa. It inhibits ATP hydrolysis by FleQ. Chanchal et al. show that FleN undergoes structural remodeling on ATP binding and that the ATPase activity of FleN is not essential for its antiactivator function. Reversible dimerization allows FleN to fine-tune the active cellular concentration of FleQ rather than exert a binary on/off effect and this ensures formation of a single flagellum.