Order-disorder-order transitions mediate the activation of cholera toxin.
Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Delta delta(coil)) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD(+), which revealed that the formation of the complex does not significantly enhance NAD(+) binding. Partial unfolding of CTA1 is further illustrated by using 4,4'-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G(s)alpha, triggering the downstream events associated with the pathophysiology of cholera.
Ampapathi
Ravi S
RS
Department of Biology and Biochemistry, University of Houston, Houston, TX, USA.
Creath
Andrea L
AL
Lou
Dianne I
DI
Craft
John W
JW
Blanke
Steven R
SR
Legge
Glen B
GB
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry; D000494:Allosteric Regulation; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002772:Cholera Toxin / Q000737:chemistry*; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D009243:NAD; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human