Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs.
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.


OBJECTIVE
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.
Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany.
Juliane
J
Düvel
Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany.
Sarina
S
Bense
Department of Biochemistry, University of Kassel, Kassel, Germany.
Stefan
S
Möller
Department of Biochemistry, University of Kassel, Kassel, Germany.
Daniela
D
Bertinetti
Biolog Life Science Institute, Bremen, Germany.
Frank
F
Schwede
Department of Chemical Biology, Helmholtz Center for Infection Research, Braunschweig, Germany.
Michael
M
Morr
Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany.
Denitsa
D
Eckweiler
Biolog Life Science Institute, Bremen, Germany.
Hans-Gottfried
HG
Genieser
Department of Cellular Proteomics, Helmholtz Center for Infection Research, Braunschweig, Germany.
Lothar
L
Jänsch
Department of Biochemistry, University of Kassel, Kassel, Germany.
Friedrich W
FW
Herberg
Department of Chemical Biology, Helmholtz Center for Infection Research, Braunschweig, Germany.
Ronald
R
Frank
Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany Susanne.Haeussler@helmholtz-hzi.de.
Susanne
S
Häussler
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016384:Consensus Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009068:Movement; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP