title: Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I.
abstract: The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Schuler
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Lübker
authors.affiliation: Molecular Graphics and Modeling Laboratory, The University of Kansas, KS 66045, USA.
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Lushington
authors.affiliation: Ben May Department of Cancer Research, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Wei-Jen
authors.initials: WJ
authors.lastname: Tang
authors.affiliation: College of Life Sciences, Nankai University, 300071 Tianjin, People's Republic of China.
authors.forename: Yuequan
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Richter
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D001667:Binding, Competitive; D001886:Bordetella pertussis / Q000201:enzymology* / Q000472:pathogenicity; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D014268:Trifluoperazine / Q000737:chemistry / Q000494:pharmacology; D014774:Virulence; D014917:Whooping Cough / Q000201:enzymology / Q000382:microbiology
chemical_list: D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; D013449:Sulfonamides; D014268:Trifluoperazine; C031938:calmidazolium; C017967:W 7; D000262:Adenylyl Cyclases; C106670:adenylyl cyclase 1