Interaction of the diguanylate cyclase YdeH of Escherichia coli with 2',(3')-substituted purine and pyrimidine nucleotides.
Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (c-di-GMP), which is degraded by specific phosphodiesterases. c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to antibiotic treatment and represent the predominant lifestyle in most chronic infections. Hence, DGCs serve as starting point for the development of novel therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2'(3')-O-(N-methylanthraniloyl) (MANT)- and 2',3'-O-(2,4,6-trinitrophenyl) (TNP)-substituted nucleotides are potent adenylyl and guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT purine and pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between tryptophan and tyrosine residues of YdeH and the MANT group of MANT-NTPs (MANT-ATP, -CTP, -GTP, -ITP, -UTP, and -XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC PleD and found high similarities in the nucleotide-binding site of PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/TNP nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into nucleotide-protein interactions.
Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Christian
C
Spangler
Volkhard
V
Kaever
Roland
R
Seifert
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011685:Purine Nucleotides / Q000235:genetics / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase