title: Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: John C
authors.initials: JC
authors.lastname: Whitney
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Whitfield
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Lindsey S
authors.initials: LS
authors.lastname: Marmont
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Yip
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
authors.forename: A Mirela
authors.initials: AM
authors.lastname: Neculai
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Yuri D
authors.initials: YD
authors.lastname: Lobsanov
authors.affiliation: the Photon Sciences Division, Brookhaven National Laboratory, Upton, New York 11973-5000, and.
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.affiliation: the Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center and McGuire Veterans Affairs Medical Center, Richmond, Virginia 23298-0678.
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Ohman
authors.affiliation: From the Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, howell@sickkids.ca.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D000464:Alginates / Q000737:chemistry*; D001426:Bacterial Proteins; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020723:Glucuronic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins; D009154:Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C085500:Alg44 protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D008565:Membrane Proteins; D037521:Virulence Factors; C062025:bis(3',5')-cyclic diguanylic acid; D020723:Glucuronic Acid; D006152:Cyclic GMP
keywords: PilZ; Pseudomonas aeruginosa (P. aeruginosa); alginate; biofilm; crystal structure; cyclic di-GMP (c-di-GMP); exopolysaccharide; isothermal titration calorimetry (ITC)
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