2.50
Hdl Handle:
http://hdl.handle.net/10033/583988
Title:
Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs
Authors:
Düvel, Juliane; Bense, Sarina; Möller, Stefan; Bertinetti, Daniela; Schwede, Frank; Morr, Michael; Eckweiler, Denitsa; Genieser, Hans-Gottfried; Jänsch, Lothar; Herberg, Friedrich W.; Frank, Ronald ( 0000-0003-2683-6511 ) ; Häussler, Susanne
Abstract:
ABSTRACT High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP 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 1990s, 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 a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be 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), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. 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 mode of growth 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 have been 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.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
Citation:
Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs 2016, 198 (1):138 Journal of Bacteriology
Journal:
Journal of Bacteriology
Issue Date:
1-Jan-2016
URI:
http://hdl.handle.net/10033/583988
DOI:
10.1128/JB.00377-15
PubMed ID:
26324453[
Additional Links:
http://jb.asm.org/lookup/doi/10.1128/JB.00377-15
Type:
Article
ISSN:
1098-5530
Appears in Collections:
publications of the departmentment of molecular bacteriology(MOBA)

Full metadata record

DC FieldValue Language
dc.contributor.authorDüvel, Julianeen
dc.contributor.authorBense, Sarinaen
dc.contributor.authorMöller, Stefanen
dc.contributor.authorBertinetti, Danielaen
dc.contributor.authorSchwede, Franken
dc.contributor.authorMorr, Michaelen
dc.contributor.authorEckweiler, Denitsaen
dc.contributor.authorGenieser, Hans-Gottfrieden
dc.contributor.authorJänsch, Lotharen
dc.contributor.authorHerberg, Friedrich W.en
dc.contributor.authorFrank, Ronalden
dc.contributor.authorHäussler, Susanneen
dc.date.accessioned2015-12-16T15:00:18Zen
dc.date.available2015-12-16T15:00:18Zen
dc.date.issued2016-01-01en
dc.identifier.citationApplication of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs 2016, 198 (1):138 Journal of Bacteriologyen
dc.identifier.issn1098-5530en
dc.identifier.pmid26324453[en
dc.identifier.doi10.1128/JB.00377-15en
dc.identifier.urihttp://hdl.handle.net/10033/583988en
dc.description.abstractABSTRACT High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP 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 1990s, 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 a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be 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), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. 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 mode of growth 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 have been 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.en
dc.relation.urlhttp://jb.asm.org/lookup/doi/10.1128/JB.00377-15en
dc.titleApplication of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifsen
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.en
dc.identifier.journalJournal of Bacteriologyen

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