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.

Members of the AAA+ (ATPase associated with various cellular activities) family of ATPases couple chemical energy derived from ATP hydrolysis for generation of mechanical force, resulting in conformational changes. The hydrolysis is brought about by highly conserved domains and motifs. The sensor I motif is critical for sensing and hydrolysis of the nucleotide. Pseudomonas aeruginosa FleQ is an ATPase that is a positive regulator of flagellar gene expression. We have determined the crystal structures of the ATPase domain of wild-type FleQ and sensor I mutants H287N and H287A in complex with ATPγS and Mg2+ to 2.4, 1.95, and 2.25 Å resolution, respectively. The structural data highlight the role of sensor I in regulating the ATPase activity. The in vitro and in vivo data demonstrate that the moderate ATPase activity of FleQ due to the presence of histidine in sensor I is essential for maintaining the monotrichous phenotype and for the rapid motility to biofilm transition.

FleN uses allostery and structural remodeling to fine-tune transcription activation by FleQ for monotrichous phenotype.