Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin.
The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.
Bauche
Cécile
C
Unité de Biochimie des Interactions Macromoléculaires, CNRS URA 2185, Institut Pasteur, 75724 Paris Cedex 15, France.
Chenal
Alexandre
A
Unité de Biochimie des Interactions Macromoléculaires, CNRS URA 2185, Institut Pasteur, 75724 Paris Cedex 15, France.
Knapp
Oliver
O
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Federal Republic of Germany.
Bodenreider
Christophe
C
Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel, CH-4056 Basel, Switzerland.
Benz
Roland
R
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Federal Republic of Germany.
Chaffotte
Alain
A
UnitédeRésonance Magnétique Nucléaire des Biomolécules, CNRS URA 2185, Institut Pasteur, F-75724 Paris, France.
Ladant
Daniel
D
Unité de Biochimie des Interactions Macromoléculaires, CNRS URA 2185, Institut Pasteur, 75724 Paris Cedex 15, France. Electronic address: ladant@pasteur.fr.
D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001886:Bordetella pertussis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D001224:Aspartic Acid; D010450:Endopeptidases; C087822:alkaline protease