High levels of the universal bacterial second messenger c-di-GMP promote the establishment of surface–attached growth in many bacteria. C-di-GMP can not only bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 90s the synthetic peptide array technique has become a powerful tool for high throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with high affinity and peptide arrays uncovered LKKALKKQTNLR as a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RxxxR) or the I-site of diguanylate cyclases (RxxD), not the charged amino acids provided the key residues of the binding sequence but two leucine residues and a glutamine residue. Those three amino acids are highly conserved across PA3740 homologs and their singular exchange to alanine reduced c-di-GMP binding within the full length protein.Importance In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains were identified. Nevertheless for several c-di-GMP receptors the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.
%0 Journal Article
%1 Düvel31082015
%A Düvel, J
%A Bense, S
%A Möller, S
%A Bertinetti, D
%A Schwede, F
%A Morr, M
%A Eckweiler, D
%A Genieser, H G
%A Jänsch, Lothar
%A Herberg, Friedrich W.
%A Frank, Ronald
%A Häussler, Susanne
%D 2015
%E Bacteriol, J
%J Journal of Bacteriology
%K haeussler
%N 1
%P 138-146
%T Application of synthetic peptide arrays to uncover c-di-GMP binding motifs
%V 198
%X High levels of the universal bacterial second messenger c-di-GMP promote the establishment of surface–attached growth in many bacteria. C-di-GMP can not only bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 90s the synthetic peptide array technique has become a powerful tool for high throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with high affinity and peptide arrays uncovered LKKALKKQTNLR as a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RxxxR) or the I-site of diguanylate cyclases (RxxD), not the charged amino acids provided the key residues of the binding sequence but two leucine residues and a glutamine residue. Those three amino acids are highly conserved across PA3740 homologs and their singular exchange to alanine reduced c-di-GMP binding within the full length protein.Importance In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains were identified. Nevertheless for several c-di-GMP receptors the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.
@article{Düvel31082015,
abstract = {High levels of the universal bacterial second messenger c-di-GMP promote the establishment of surface–attached growth in many bacteria. C-di-GMP can not only bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 90s the synthetic peptide array technique has become a powerful tool for high throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with high affinity and peptide arrays uncovered LKKALKKQTNLR as a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RxxxR) or the I-site of diguanylate cyclases (RxxD), not the charged amino acids provided the key residues of the binding sequence but two leucine residues and a glutamine residue. Those three amino acids are highly conserved across PA3740 homologs and their singular exchange to alanine reduced c-di-GMP binding within the full length protein.Importance In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains were identified. Nevertheless for several c-di-GMP receptors the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.},
added-at = {2015-09-09T11:57:41.000+0200},
author = {Düvel, J and Bense, S and Möller, S and Bertinetti, D and Schwede, F and Morr, M and Eckweiler, D and Genieser, H G and Jänsch, Lothar and Herberg, Friedrich W. and Frank, Ronald and Häussler, Susanne},
biburl = {https://www.bibsonomy.org/bibtex/2ffaf7cc7d035424f8329ebe23e79d8d3/haeussler},
editor = {Bacteriol, J},
interhash = {45a33b95818a60e156c615b66551fa35},
intrahash = {ffaf7cc7d035424f8329ebe23e79d8d3},
journal = {Journal of Bacteriology},
keywords = {haeussler},
number = 1,
pages = {138-146},
pubmedurl = {http://www.ncbi.nlm.nih.gov/pubmed/26324453},
timestamp = {2016-01-11T12:05:43.000+0100},
title = {Application of synthetic peptide arrays to uncover c-di-GMP binding motifs},
volume = 198,
year = 2015
}