title: Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa.
abstract: High cellular concentrations of bis-(3',5')-cyclic dimeric guanosine mono-phosphate (c-di-GMP) regulate a diverse range of phenotypes in bacteria including biofilm development. The opportunistic pathogen Pseudomonas aeruginosa produces the PEL polysaccharide to form a biofilm at the air-liquid interface of standing cultures. Among the proteins required for PEL polysaccharide production, PelD has been identified as a membrane-bound c-di-GMP-specific receptor. In this work, we present the x-ray crystal structure of a soluble cytoplasmic region of PelD in its apo and c-di-GMP complexed forms. The structure of PelD reveals an N-terminal GAF domain and a C-terminal degenerate GGDEF domain, the latter of which binds dimeric c-di-GMP at an RXXD motif that normally serves as an allosteric inhibition site for active diguanylate cyclases. Using isothermal titration calorimetry, we demonstrate that PelD binds c-di-GMP with low micromolar affinity and that mutation of residues involved in binding not only decreases the affinity of this interaction but also abrogates PEL-specific phenotypes in vivo. Bioinformatics analysis of the juxtamembrane region of PelD suggests that it contains an α-helical stalk region that connects the soluble region to the transmembrane domains and that similarly to other GAF domain containing proteins, this region likely forms a coiled-coil motif that mediates dimerization. PelD with Alg44 and BcsA of the alginate and cellulose secretion systems, respectively, collectively constitute a group of c-di-GMP receptors that appear to regulate exopolysaccharide assembly at the protein level through activation of their associated glycosyl transferases.
authors.affiliation: Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: John C
authors.initials: JC
authors.lastname: Whitney
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Colvin
authors.forename: Lindsey S
authors.initials: LS
authors.lastname: Marmont
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Parsek
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D018441:Biofilms; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; C064841:exopolysaccharide, Pseudomonas; C062025:bis(3',5')-cyclic diguanylic acid; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D006152:Cyclic GMP