Sensor I Regulated ATPase Activity of FleQ Is Essential for Motility to Biofilm Transition in Pseudomonas aeruginosa

Abstract

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.

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

“…Besides the movement of the two subdomains, there are substantial conformational changes in the residues that are otherwise involved in interaction with the nucleotide in the ATPγS-bound structure of FleQ AAA+ ( 9 ). K180 of the Walker A motif, which interacts with the phosphates of the nucleotide, exhibits altered conformation in the FleN-bound structure.…”

“…In the crystal structure of FleQ-ATPγS, the Walker B residue E246, present in the DEXX motif, interacts with sensor I H287 and a catalytic water molecule that makes a nucleophilic attack on the γ-phosphate of ATP for hydrolysis. This state is the catalytically competent state of the protein ( 9 ). However, in the FleN-bound form, due to the movement of the FleQ subdomains closer to each other causing the release of ATP, the side chain of E246 flips (90° rotation) and interacts with N202, also called the Asn switch ( Fig.…”

“…Unlike in the case of negative regulators, the NTD of FleQ does not inhibit its ATPase activity; instead, it enables the formation of the functional hexameric form ( 15 , 16 ). In addition, we have earlier demonstrated that FleQ sensor I is a histidine residue that is unique in place of highly conserved asparagine found in the majority of the other family members ( 9 ). This ensures that, although constitutive, the ATPase activity is depleted compared to the other AAA+ proteins that contain a conserved Asn in sensor I.…”

“…The central domain is further composed of two subdomains, the α/β subdomain at the N terminus followed by the α-helical subdomain toward the C terminus. The nucleotide binds at the cleft between the two subdomains ( 9 ). In addition, the central domain contains two surface-exposed loops L1 and L2.…”

The evolution of virulence inPseudomonas aeruginosaduring chronic wound infection

“…The mutations in rpoN, fleQ and pilR all lead to amino acid changes within highly conserved domains or residues directly involved in the activity of their respective protein products [73][74][75]. The gene product of rpoN, RNA polymerase factor σ 54 , regulates a wide variety of functions in P. aeruginosa, including the rhl QS system, flagellin and pilin production, which play important roles in motility, surface attachment and biofilm formation [54,55,[60][61][62][63].…”