FleN (PA1454) is a 280-residue cytoplasmic MinD/ParA-family (FlhG-like) P-loop ATPase that sets flagellar number in Pseudomonas aeruginosa by acting as an anti-activator of FleQ, the sigma54-dependent AAA+ enhancer-binding protein at the top of the flagellar transcription hierarchy. Binding of ATP at its deviant Walker A motif remodels FleN into a nucleotide-sandwich homodimer; the ATP-bound dimer binds the FleQ AAA+ domain, allosterically blocks ATP binding and hydrolysis by FleQ and prevents productive FleQ oligomerization, so dampening FleQ-dependent expression of flagellar genes. FleN itself has weak intrinsic ATPase activity, and hydrolysis is proposed to release FleQ and allow basal flagellar gene expression. Loss of fleN causes up-regulation of FleQ-dependent flagellar genes, three to six polar flagella per cell and loss of swimming and chemotactic motility. With FleQ and c-di-GMP, FleN also participates in the regulation of biofilm (pel) genes, bridging DNA-bound FleQ to bend the pelA promoter.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA ATP binding from PAINT node PTN000344128, whose only seed is the FleN PseudoCAP IDA row (PMID:10629180) itself. Reason: ATP binding is correct for FleN and for the MinD/ParA/FlhG clade under PTN000344128: FleN crystal structures with an ATP analog show the nucleotide bound at the Walker A motif and ATP binding drives dimerization (PMID:28065505, PMID:34669473), and the deviant Walker A/P-loop is conserved across the family (PIRSF003092, IPR025501). The IBD conclusion is therefore sound in substance and FleN appearing in its own WITH/FROM is expected, not circular. The provenance problem is that the sole seed row is not direct experimental evidence (the 2000 paper inferred ATP binding from a sequence motif, see the IDA row); the node should be re-seeded with genuine experimental evidence (PMID:28065505 for FleN, or ATP-binding data for E. coli MinD). Recorded as a GO_CENTRAL source defect; the IBA on FleN itself is accepted. Propagation Review Root cause: NO FAILURE CORE Failure modes: SOURCE EVIDENCE WEAK Sources checked: UniProtKB:G3XD64 SOURCE WEAK OR INFERRED The sole IBD seed is the PseudoCAP IDA row citing PMID:10629180, whose abstract reports an N-terminal ATP-binding sequence motif rather than a binding assay (full text not read). The claim is nevertheless true (FleN ATP-analog crystal structures, PMID:28065505), so the transferred term is correct; only the seed provenance is weak. Supporting Evidence: PMID:28065505 Crystal structures of FleN in the apo form (1.66 Γ
) and in complex with Ξ²,Ξ³-imidoadenosine 5'-triphosphate (1.55 Γ
) reveal that it undergoes drastic conformational changes on ATP binding to attain a structure capable of dimerization. PMID:34669473 The two ATPΞ³S molecules are present at the interface of the FleN dimer such that each nucleotide is bound to one monomer and the binding is stabilized by residues K19, E153, and T155 of the adjacent monomer |
| GO:0005524 ATP binding | IDA PMID:10629180 fleN, a gene that regulates flagellar number in Pseudomonas ... | ACCEPT | Summary: PseudoCAP IDA ATP binding citing the original fleN identification paper; the claim is true but the cited paper inferred it from an N-terminal ATP-binding sequence motif, not an assay. Reason: The term is correct and central to FleN mechanism: ATP binding at the deviant Walker A motif remodels FleN into the dimer that antagonizes FleQ (PMID:28065505, PMID:34669473, PMID:38051770). However, the abstract of PMID:10629180 reports only an ORF "with an ATP-binding motif at its N terminus", and the same group described FleN as "a putative ATP/GTP binding protein" a year later (PMID:11673434), which indicates no nucleotide-binding assay was performed in 2000. The full text of PMID:10629180 is not in our cache, so this is PROBABLE rather than CONFIRMED, but the evidence for the row is a sequence motif (ISM/ISS), not IDA. The claim is accepted on the strength of later direct structural evidence; the evidence code/reference is recorded as a PseudoCAP source defect. Supporting Evidence: PMID:10629180 that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found PMID:11673434 Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein PMID:28065505 Crystal structures of FleN in the apo form (1.66 Γ
) and in complex with Ξ²,Ξ³-imidoadenosine 5'-triphosphate (1.55 Γ
) reveal that it undergoes drastic conformational changes on ATP binding to attain a structure capable of dimerization. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: IBA cytosol from the MinD/FlhG clade node, seeded from E. coli MinD. Reason: FleN is a soluble protein without transmembrane segments that acts on FleQ bound at promoter DNA; a fluorescent-protein fusion of FleN was evenly distributed in P. aeruginosa cells, consistent with a cytosolic location. Cytosol is as specific as the evidence allows. Supporting Evidence: PMID:36622167 FleN was equally distributed in the cells independent of the terminus to which fluorescent protein was attached PMID:22581773 at these sites causing the intervening DNA to bend |
| GO:0009898 cytoplasmic side of plasma membrane | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA cytoplasmic side of plasma membrane inherited from E. coli MinD, which binds the membrane through a C-terminal amphipathic membrane-targeting helix. Reason: The membrane association of MinD (the sole WITH/FROM donor) is mediated by its C-terminal membrane-targeting sequence and is integral to MinCDE oscillation. For FleN there is no evidence of membrane association; a fluorescent fusion was diffusely distributed and FleN acts by binding FleQ, including FleQ bound at promoter DNA. The FleN C-terminal region that in homologs mediates membrane binding is instead used for FleQ contact. The location is therefore not supported for FleN, although it may hold for membrane-targeted FlhG orthologs in other species. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:P0AEZ3 SUPPORTS SOURCE BUT NOT TARGET E. coli MinD binds the inner membrane through its C-terminal membrane-targeting sequence as part of MinCDE oscillation; FleN acts as a soluble FleQ anti-activator and a FleN fluorescent fusion is diffusely distributed (PMID:36622167). Supporting Evidence: PMID:36622167 FleN was equally distributed in the cells independent of the terminus to which fluorescent protein was attached PMID:34669473 This region is disordered in the FleN dimer and has been implicated in membrane binding in homologous proteins |
| GO:0016887 ATP hydrolysis activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA ATP hydrolysis activity from the MinD/ParA clade, seeded from E. coli MinD. Reason: FleN has measurable, weak intrinsic ATPase activity; the Walker A K19A mutant hydrolyzes ATP at about 16% of wild type and D48A is an ATP-binding but hydrolysis-incompetent mutant. Hydrolysis is proposed to dissociate the FleN dimer from FleQ and allow basal flagellar gene expression, so this is a genuine activity of FleN, though anti-activation of FleQ requires ATP binding rather than hydrolysis. Supporting Evidence: PMID:38051770 was found to no longer dimerize or hydrolyze ATP efficiently (at about 16% relative to wild-type FleN) PMID:38051770 ATP-binding but hydrolysis-incompetent FleND48A mutant PMID:34669473 residual activity is similar to the ATPase activity of FleN |
| GO:0042030 ATPase inhibitor activity | IDA PMID:34669473 The antiactivator FleN uses an allosteric mechanism to regul... | NEW | Summary: Proposed new MF capturing the FleQ anti-activator activity of FleN. Reason: FleN binds the AAA+ domain of FleQ and inhibits its ATPase activity in vitro (wild-type FleN abolishes FleQ ATPase activity; FleQ-interface mutants inhibit less well and fail to complement the multiflagellate phenotype). This is the direct molecular basis of anti-activation and is the most informative MF available; GO has no anti-activator or transcription-activator-inhibitor term for a non-DNA-binding protein that inhibits a bacterial enhancer-binding protein. Supporting Evidence: PMID:34669473 FleN WT completely abolishes the ATPase activity of FleQFL PMID:34669473 The data reveal that FleN allosterically prevents ATP binding to FleQ. PMID:28065505 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. |
| GO:1902209 negative regulation of bacterial-type flagellum assembly | IMP PMID:10629180 fleN, a gene that regulates flagellar number in Pseudomonas ... | NEW | Summary: Proposed new BP for the flagellar number control role of FleN. Reason: FleN itself performs the regulatory step (it binds and inhibits FleQ, the activator of class II flagellar genes). Loss of fleN up-regulates FleQ-dependent flagellar promoters and yields three to six polar flagella per cell, complemented by plasmid fleN; FleQ-interface mutants of FleN fail to restore monotrichy. This is the core biological role of FleN and is not yet annotated. Supporting Evidence: PMID:10629180 the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type PMID:34669473 motility and transcription defects in vivo and multiflagellate phenotype |
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Download this section (compressed HTML)Q: Should GO provide a term for inhibition of a bacterial enhancer-binding protein (anti-activator activity) by a non-DNA-binding partner, distinct from generic ATPase inhibitor activity?
Q: Can the PAINT IBD for ATP binding at PTN000344128 be re-seeded with direct experimental evidence (FleN PDB 5J1J or E. coli MinD) before the PseudoCAP IDA row is corrected?
Experiment: Measure FleN ATP binding affinity (ITC or fluorescent nucleotide) and ATPase kinetics in the presence and absence of FleQ and c-di-GMP to establish how hydrolysis times release of FleQ.
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