Goettle, Martin and Dove, Stefan and Steindel, Phillip and Shen, Yuequan and Tang, Wei-Jen and Geduhn, Jens and Koenig, Burkhard and Seifert, Roland (2007) Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent Nucleotides. MOLECULAR PHARMACOLOGY, 72 (3). pp. 526-535. ISSN 0026-895X, 1521-0111
Full text not available from this repository. (Request a copy)Abstract
The calmodulin ( CaM)- dependent adenylyl cyclase ( AC) toxin from Bordetella pertussis ( CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2 ', 3 '- Nmethylanthraniloyl ( MANT)-, anthraniloyl- and trinitrophenyl ( TNP)- substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5- to 170- fold increased potency of nucleotides. K i values of 3 ' MANT- 2 ' dATP and 2 ' MANT- 3 ' d- ATP in the AC activity assay using Mn2 (+) were 220 and 340 nM, respectively. Natural nucleoside 5 'triphosphates, guanine-, hypoxanthine- and pyrimidine- MANTand TNP nucleotides and di- MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer ( FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K (d) values of 300 nM for 3 ' MANT- 2 ' d- ATP and 400 nM for 2 ' MANT- 3 ' d- ATP. Fluorescence experiments and docking approaches indicate that the MANT- and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM- dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to lowaffinity MANT nucleotides, even low- affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | BACILLUS-ANTHRACIS; STRUCTURAL BASIS; TOXIN; INHIBITION; CALMODULIN; ANALOGS; INFECTION; BINDING; PURINE; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy Chemistry and Pharmacy > Institute of Pharmacy > Alumni or Retired Professors > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) Chemistry and Pharmacy > Institut für Organische Chemie Chemistry and Pharmacy > Institut für Organische Chemie > Lehrstuhl Prof. Dr. Burkhard König |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 01 Dec 2020 12:51 |
| Last Modified: | 01 Dec 2020 12:51 |
| URI: | https://pred.uni-regensburg.de/id/eprint/32309 |
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