Interaction of calcium with Bordetella pertussis adenylate cyclase toxin. Characterization of multiple calcium-binding sites and calcium-induced conformational changes.
The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.
Rose
T
T
Laboratoire de Résonance Magnétique Nucléaire, Institut Pasteur, Paris, France.
Sebo
P
P
Bellalou
J
J
Ladant
D
D
D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001886:Bordetella pertussis / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology; D005798:Genes, Bacterial; D006461:Hemolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D017433:Protein Structure, Secondary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D002118:Calcium