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A cyclic-di-GMP receptor required for bacterial exopolysaccharide production.

Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S - Mol. Microbiol. (2007)

Bottom Line: Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide.Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP.The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

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Affinity of PelD for c-di-GMP. MBP-PelD is chemically cross-linked on gold surface and probed with either c-di-GMP at 5 μM (brown), 10 μM (purple) or 20 μM (cyan) or GTP at 5 μM (yellow), 10 μM (pink) or 20 μM (blue) and washed away at times indicated by the arrows. A. MBP-PelD. B. MBP-PelD (R367A). C. MBP-PelD (D370A). D. MBP-PelD (R402A).
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fig04: Affinity of PelD for c-di-GMP. MBP-PelD is chemically cross-linked on gold surface and probed with either c-di-GMP at 5 μM (brown), 10 μM (purple) or 20 μM (cyan) or GTP at 5 μM (yellow), 10 μM (pink) or 20 μM (blue) and washed away at times indicated by the arrows. A. MBP-PelD. B. MBP-PelD (R367A). C. MBP-PelD (D370A). D. MBP-PelD (R402A).

Mentions: We determined the affinity of PelD for c-di-GMP using the surface plasmon resonance technique. The MBP-PelD or point mutant variants were chemically cross-linked onto gold surface. Either c-di-GMP or GTP at 5, 10 or 20 μM was passed over the protein-coated surface. An interaction between the protein and ligand results in a mass change of the complex that is detectable by a change in the resonance frequency of the gold surface and is read out as relative units (RU). MBP-PelD was able to bind c-di-GMP with a dissociation constant (KD) of 1 μM (Fig. 4A). Point mutants (R367A, D370A or R402A) were not able to bind c-di-GMP (Fig. 4B–D respectively). None of the MBP-PelD or point mutants bound GTP suggesting that PelD is not a diguanylate cyclase. The theoretical maximum response is proportional to the ratio of the molecular weight of the ligand and the protein and the number of binding sites for the ligand. As 6000 RU of protein was cross-linked onto each spot of the chip, the maximum response would be 50 RU for one c-di-GMP binding site and 100 RU for two c-di-GMP binding site. As the protein is chemically linked, it is very likely oriented in many directions relative to the surface and thus only a fraction, typically half, of the PelD would be available to bind c-di-GMP. The amount of response for MBP-PelD that approaches saturation is approximately 25 RU suggesting that there is one c-di-GMP binding site per PelD protein.


A cyclic-di-GMP receptor required for bacterial exopolysaccharide production.

Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S - Mol. Microbiol. (2007)

Affinity of PelD for c-di-GMP. MBP-PelD is chemically cross-linked on gold surface and probed with either c-di-GMP at 5 μM (brown), 10 μM (purple) or 20 μM (cyan) or GTP at 5 μM (yellow), 10 μM (pink) or 20 μM (blue) and washed away at times indicated by the arrows. A. MBP-PelD. B. MBP-PelD (R367A). C. MBP-PelD (D370A). D. MBP-PelD (R402A).
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Related In: Results  -  Collection

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fig04: Affinity of PelD for c-di-GMP. MBP-PelD is chemically cross-linked on gold surface and probed with either c-di-GMP at 5 μM (brown), 10 μM (purple) or 20 μM (cyan) or GTP at 5 μM (yellow), 10 μM (pink) or 20 μM (blue) and washed away at times indicated by the arrows. A. MBP-PelD. B. MBP-PelD (R367A). C. MBP-PelD (D370A). D. MBP-PelD (R402A).
Mentions: We determined the affinity of PelD for c-di-GMP using the surface plasmon resonance technique. The MBP-PelD or point mutant variants were chemically cross-linked onto gold surface. Either c-di-GMP or GTP at 5, 10 or 20 μM was passed over the protein-coated surface. An interaction between the protein and ligand results in a mass change of the complex that is detectable by a change in the resonance frequency of the gold surface and is read out as relative units (RU). MBP-PelD was able to bind c-di-GMP with a dissociation constant (KD) of 1 μM (Fig. 4A). Point mutants (R367A, D370A or R402A) were not able to bind c-di-GMP (Fig. 4B–D respectively). None of the MBP-PelD or point mutants bound GTP suggesting that PelD is not a diguanylate cyclase. The theoretical maximum response is proportional to the ratio of the molecular weight of the ligand and the protein and the number of binding sites for the ligand. As 6000 RU of protein was cross-linked onto each spot of the chip, the maximum response would be 50 RU for one c-di-GMP binding site and 100 RU for two c-di-GMP binding site. As the protein is chemically linked, it is very likely oriented in many directions relative to the surface and thus only a fraction, typically half, of the PelD would be available to bind c-di-GMP. The amount of response for MBP-PelD that approaches saturation is approximately 25 RU suggesting that there is one c-di-GMP binding site per PelD protein.

Bottom Line: Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide.Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP.The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

Show MeSH