title: Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: clues from the structure of the catalytic domain.
abstract: Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.
authors.affiliation: Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy ; Istituto Pasteur - Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Giardina
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.forename: Silvia
authors.initials: S
authors.lastname: Fernicola
authors.forename: Stefano
authors.initials: S
authors.lastname: Franceschini
authors.forename: Serena
authors.initials: S
authors.lastname: Rinaldo
authors.forename: Valentina
authors.initials: V
authors.lastname: Stelitano
authors.forename: Francesca
authors.initials: F
authors.lastname: Cutruzzolà
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP