Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases.
Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.
Vougier
Stéphanie
S
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, EA 3106, IFR 117, Université Paris 7, Denis Diderot, 2 place Jussieu, Paris 75005, France.
Mary
Jean
J
Dautin
Nathalie
N
Vinh
Joëlle
J
Friguet
Bertrand
B
Ladant
Daniel
D
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D019921:Biotinylation; D001886:Bordetella pertussis / Q000201:enzymology*; D039481:CD11b Antigen / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007477:Ions; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D051150:Methionine Sulfoxide Reductases; D008840:Microfilament Proteins; D009504:Neutrophils / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010587:Phagocytosis; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014157:Transcription Factors; D020122:tert-Butylhydroperoxide / Q000737:chemistry
D039481:CD11b Antigen; D018821:CD18 Antigens; D002147:Calmodulin; D007477:Ions; D008840:Microfilament Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014157:Transcription Factors; D020122:tert-Butylhydroperoxide; D008715:Methionine; D000242:Cyclic AMP; D010088:Oxidoreductases; C120376:MSRB2 protein, human; D051150:Methionine Sulfoxide Reductases; C488525:MsrA protein, rat; C027219:methionine sulfoxide reductase; D000262:Adenylyl Cyclases; D010100:Oxygen; C013111:methionine sulfoxide