title: Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system.
abstract: The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide. This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs). We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively. Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system. We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6. Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT. These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF. Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity. These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo.
authors: Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80220, USA.
authors: M G
authors: MG
authors: Jobling
authors: R K
authors: RK
authors: Holmes
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics; D020816:Amino Acid Motifs; D001886:Bordetella pertussis / Q000201:enzymology / Q000235:genetics; D002772:Cholera Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D000089982:ADP-Ribosylation Factor 6; D010455:Peptides; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D000262:Adenylyl Cyclases