Structural studies of the cholera toxin catalytic subunit

Vibrio cholerae causes devastating diarrhea when it infects the human intestine. The primary virulence factor for the disease is cholera toxin (CT), a heterohexameric protein toxin that ADP-ribosylates the human signaling protein GSalpha. To form the enzymatically active toxic fragment, pro-enzyme CT undergoes limited proteolysis and disulfide bond reduction. Reduction occurs in the ER via the reductase/chaperone protein disulfide isomerase (PDI), an interaction which is further explored in this work. Without a chaperone, activational modifications render CT unstable in solution, preventing a structural understanding of conformational changes resulting from toxin activation and substrate binding.; Crystal structures of an A-subunit variant, Y30S, which requires no activational modifications for full activity, represent the first structures of active CT. Differences between CTY30S and wild-type, proenzyme CT are observed only in two A-subunit loop regions. The activation loop (residues 25-36) is disordered in CTY30S, while the active-site loop (residues 47-56) displays varying degrees of order, suggesting that activation loop disorder predisposes the active-site loop to be more flexible than in unactivated wild-type CT. A model shows how the activational modifications could be communicated to the active site.; CTA1 is allosterically activated by interaction with human ADP-ribosylation factors (ARFs). Crystal structures of a CTA1:ARF6-GTP complex reveal that the extensive toxin:ARF-GTP interface mimics ARF-GTP recognition of normal cellular protein partners, suggesting that the toxin exploits promiscuous binding properties of ARFs. Upon binding the human protein activator, toxin activation is accomplished via dramatic conformational changes in CTA1 loop regions. The activation loop rearranges to contact ARF6-GTP as an amphipathic helix, which forces the active-site loop out of the active site. The new active-site loop position generates a possible GSalpha, binding surface, one which may only be available during interaction with ARF-GTP.; Despite active-site accessibility in certain CTY30S crystal forms, soaking CTY30S crystals with massive concentrations of NAD+ resulted in poor quality and discontinuous maps of a fragment of an NAD+ molecule in the active site. However, crystals of the CTA1:ARF6-GTP complex soaked with the substrate yielded strong, continuous density that gives the first glimpse at the NAD+-binding mode of the toxin.