title: Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.
abstract: Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.
authors.affiliation: Departamento de Física e Ciência Interdisciplinar, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos 13563-120, SP, Brazil;
authors.forename: Bruno Y
authors.initials: BY
authors.lastname: Matsuyama
authors.affiliation: Department of Structural Biology and Chemistry, Unité G5 Biologie Structurale de la Sécrétion Bactérienne and UMR 3528, CNRS, Institut Pasteur, 75015 Paris, France; Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853;
authors.forename: Petya V
authors.initials: PV
authors.lastname: Krasteva
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195.
authors.forename: Claudine
authors.initials: C
authors.lastname: Baraquet
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195 csh5@uw.edu hs293@cornell.edu mvasnavarro@ifsc.usp.br.
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Harwood
authors.affiliation: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; csh5@uw.edu hs293@cornell.edu mvasnavarro@ifsc.usp.br.
authors.forename: Holger
authors.initials: H
authors.lastname: Sondermann
authors.affiliation: Departamento de Física e Ciência Interdisciplinar, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos 13563-120, SP, Brazil; csh5@uw.edu hs293@cornell.edu mvasnavarro@ifsc.usp.br.
authors.forename: Marcos V A S
authors.initials: MV
authors.lastname: Navarro
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018441:Biofilms / Q000187:drug effects*; D002151:Calorimetry; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D012996:Solutions; D013696:Temperature; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; C108098:FleQ protein, Pseudomonas aeruginosa; D050505:Mutant Proteins; D012996:Solutions; D015534:Trans-Activators; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP
keywords: enhancer binding protein; flagella; gene expression; structure