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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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Secondary structure prediction and consensus sequence of PelD and PleD. A. Secondary structure predication was made using the web-based ProteinPredict program hosted at http://www.predictprotein.org/ for PelD and PleD. The confidence level of the prediction for each amino acid is located below the prediction. α-Helix and β-sheet elements predicted with a confidence level of greater than 5 is depicted on the diagram. Conserved residues for PelD and PleD are shown above the diagram and residues required for binding are shown in red. RxxD motif is indicated by the red box and GGEEF motif is in purple and is underlined. B. Consensus sequence of PelD and PleD was based on Phyre alignment of 20 and 100 homologues respectively. Residues required for c-di-GMP binding is indicated in red, GGEEF motif is in purple and is underlined and other conserved residues are in green. RxxD motif is indicated by the red box.
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fig05: Secondary structure prediction and consensus sequence of PelD and PleD. A. Secondary structure predication was made using the web-based ProteinPredict program hosted at http://www.predictprotein.org/ for PelD and PleD. The confidence level of the prediction for each amino acid is located below the prediction. α-Helix and β-sheet elements predicted with a confidence level of greater than 5 is depicted on the diagram. Conserved residues for PelD and PleD are shown above the diagram and residues required for binding are shown in red. RxxD motif is indicated by the red box and GGEEF motif is in purple and is underlined. B. Consensus sequence of PelD and PleD was based on Phyre alignment of 20 and 100 homologues respectively. Residues required for c-di-GMP binding is indicated in red, GGEEF motif is in purple and is underlined and other conserved residues are in green. RxxD motif is indicated by the red box.

Mentions: A Pfam search demonstrated that PelD lacked a recognizable PilZ domain in contrast to the eight other P. aeruginosa genes that encode PilZ domains. Secondary structure of PelD predicted by ProteinPredict (Rost et al., 2004) suggest that the PelD protein consists of alternating α-helix–β-sheet–α-helix–β-sheet–α-helix that is distinct from the β-barrel fold of PilZ domains (Fig. 5A). Another known c-di-GMP-binding domain is the I-site of diguanylate cyclases that controls feedback inhibition as demonstrated for Caulobacter crescentus PleD (Chan et al., 2004). The I-site consists of an RxxD motif that is invariably located nine and six amino acids from the GGEEF (Christen et al., 2006). Despite a low level of sequence similarity between PelD and PleD, we sought to determine whether there are any secondary structural similarities between these two proteins. Secondary structure predicted by ProteinPredict produced results that matched the known structure determined by crystallography (Fig. 5) (Chan et al., 2004). An important component of this computational analysis is the confidence level assigned for each of the predicted elements ranging from 0 to 9 with 9 being the greatest confidence of the prediction. Using alignment tools at Phyre web server (http://www.sbg.bio.ic.ac.uk/phyre) (Kelley et al., 2000), the conserved residues in PleD for the top 100 homologues yielded a number of residues including the catalytic GGEEF motif and the RxxD I-site and allow the identification of conserved identical and similar residues (Fig. 5B). Mapping these residues on the crystal structure of the PleD revealed that the GGEEF motif and all other conserved residues map to one face of the diguanylate cyclase domain, whereas the RxxD motif is on the other face (Fig. S3). Comparison of the PelD and PleD secondary structures suggests that they have similar domain fold consisting of alternating α-helix–β-sheet–α-helix–β-sheet–α-helix (Fig. 5B). Furthermore, the RxxD residues required for binding c-di-GMP are present in both proteins at similar position within the domain (Fig. 5B). Despite these similarities, PelD is obviously lacking the GG(D/E)F motif of diguanylate cyclases as well as other conserved residues. Furthermore, the equivalent of PelD R402 is much less conserved in PleD-like homologues. Together these results suggest the potential of a wide array of proteins that can utilize a simple RxxD c-di-GMP-binding motif to regulate the activity of other portions of the 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)

Secondary structure prediction and consensus sequence of PelD and PleD. A. Secondary structure predication was made using the web-based ProteinPredict program hosted at http://www.predictprotein.org/ for PelD and PleD. The confidence level of the prediction for each amino acid is located below the prediction. α-Helix and β-sheet elements predicted with a confidence level of greater than 5 is depicted on the diagram. Conserved residues for PelD and PleD are shown above the diagram and residues required for binding are shown in red. RxxD motif is indicated by the red box and GGEEF motif is in purple and is underlined. B. Consensus sequence of PelD and PleD was based on Phyre alignment of 20 and 100 homologues respectively. Residues required for c-di-GMP binding is indicated in red, GGEEF motif is in purple and is underlined and other conserved residues are in green. RxxD motif is indicated by the red box.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170427&req=5

fig05: Secondary structure prediction and consensus sequence of PelD and PleD. A. Secondary structure predication was made using the web-based ProteinPredict program hosted at http://www.predictprotein.org/ for PelD and PleD. The confidence level of the prediction for each amino acid is located below the prediction. α-Helix and β-sheet elements predicted with a confidence level of greater than 5 is depicted on the diagram. Conserved residues for PelD and PleD are shown above the diagram and residues required for binding are shown in red. RxxD motif is indicated by the red box and GGEEF motif is in purple and is underlined. B. Consensus sequence of PelD and PleD was based on Phyre alignment of 20 and 100 homologues respectively. Residues required for c-di-GMP binding is indicated in red, GGEEF motif is in purple and is underlined and other conserved residues are in green. RxxD motif is indicated by the red box.
Mentions: A Pfam search demonstrated that PelD lacked a recognizable PilZ domain in contrast to the eight other P. aeruginosa genes that encode PilZ domains. Secondary structure of PelD predicted by ProteinPredict (Rost et al., 2004) suggest that the PelD protein consists of alternating α-helix–β-sheet–α-helix–β-sheet–α-helix that is distinct from the β-barrel fold of PilZ domains (Fig. 5A). Another known c-di-GMP-binding domain is the I-site of diguanylate cyclases that controls feedback inhibition as demonstrated for Caulobacter crescentus PleD (Chan et al., 2004). The I-site consists of an RxxD motif that is invariably located nine and six amino acids from the GGEEF (Christen et al., 2006). Despite a low level of sequence similarity between PelD and PleD, we sought to determine whether there are any secondary structural similarities between these two proteins. Secondary structure predicted by ProteinPredict produced results that matched the known structure determined by crystallography (Fig. 5) (Chan et al., 2004). An important component of this computational analysis is the confidence level assigned for each of the predicted elements ranging from 0 to 9 with 9 being the greatest confidence of the prediction. Using alignment tools at Phyre web server (http://www.sbg.bio.ic.ac.uk/phyre) (Kelley et al., 2000), the conserved residues in PleD for the top 100 homologues yielded a number of residues including the catalytic GGEEF motif and the RxxD I-site and allow the identification of conserved identical and similar residues (Fig. 5B). Mapping these residues on the crystal structure of the PleD revealed that the GGEEF motif and all other conserved residues map to one face of the diguanylate cyclase domain, whereas the RxxD motif is on the other face (Fig. S3). Comparison of the PelD and PleD secondary structures suggests that they have similar domain fold consisting of alternating α-helix–β-sheet–α-helix–β-sheet–α-helix (Fig. 5B). Furthermore, the RxxD residues required for binding c-di-GMP are present in both proteins at similar position within the domain (Fig. 5B). Despite these similarities, PelD is obviously lacking the GG(D/E)F motif of diguanylate cyclases as well as other conserved residues. Furthermore, the equivalent of PelD R402 is much less conserved in PleD-like homologues. Together these results suggest the potential of a wide array of proteins that can utilize a simple RxxD c-di-GMP-binding motif to regulate the activity of other portions of the 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