| This study used a bacterial two-hybrid system in which reconstitution of CAMP-dependent signal transduction in an Escherichia coli cyaA mutant is dependent on interactions between the CTA1 and ARF6 domains of T25-ARF6 and CTA1-T18 fusion proteins that contain T18 and T25 polypeptides derived from Bordetella pertussis adenylate cyclase. We identified twenty-one single-amino-acid-substitution variants of T25-ARF6 that decreased its ability to interact with CTA1-T18 in the bacterial two-hybrid system. Representative ARF6 variants with corresponding substitutions were purified and characterized. Fifteen of these variants had ≥40% of the GTP-binding activity of native ARF6, but most were much less active than native ARF6 in activating the ADP-ribosyltransferase activity of CTAI. We also identified suppressor mutations that restored the ability of inactive variants of CTA1 and Arf6 to interact with each other. Additionally, we characterized CTA1 variants that retained basal levels of enzymatic activity but lacked the ability to be activated by Arf6.; During the course of the research presented here the structure of a CTA1::Arf6-GTP complex was determined by X-ray crystallography (180). Examination of published structures for ARF6-GTP and CTA1::ARF6-GTP revealed that the substitutions in ARF6 described above were located in switch region I, switch region II, the interswitch region or the C-terminal alpha-helix and often were not surface exposed. Many of the corresponding residues in wild type ARF6 did not make direct contacts with CTA1 in the CTA1::ARF6-GTP complex, although they were close to such contact residues in the three dimensional structure of ARF6. Taken together, our results suggest that single-amino-acid substitutions for residues that do not make direct contacts with CTA1 can prevent both formation of CTA1::ARF6-GTP complexes and allosteric activation of CTA1 by causing local distortions in ARF6 motifs that would otherwise mediate interactions with CTA1. The results of our study are complementary to this recent structure and provide additional insights about structural features of CTA1 and Arf6 that are important for their physical interactions and biological functions. |
