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Open AccessArticle

A Mutational Analysis of Residues in Cholera Toxin A1 Necessary for Interaction with Its Substrate, the Stimulatory G Protein Gsα

Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver, 12800 E 19th Ave, Aurora, CO 80045, USA
Department of Biology, Metropolitan State University of Denver, P.O. Box 173362, CB 53, Denver, CO 80217, USA
Atila Biosystems Inc., 740 Sierra Vista Ave, Unit E, Mountain View, CA 94043, USA
Author to whom correspondence should be addressed.
Academic Editor: Ken Teter
Toxins 2015, 7(3), 919-935;
Received: 18 December 2014 / Revised: 27 February 2015 / Accepted: 4 March 2015 / Published: 18 March 2015
(This article belongs to the Special Issue The Cell Biology of Toxins and Effector Proteins from Vibrio cholerae)
Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging “active-site” and “activation” loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small substrate predicted to fit into the CTA1 active site). Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα. View Full-Text
Keywords: cholera toxin; Gs alpha; ADP-ribosylation cholera toxin; Gs alpha; ADP-ribosylation
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Jobling, M.G.; Gotow, L.F.; Yang, Z.; Holmes, R.K. A Mutational Analysis of Residues in Cholera Toxin A1 Necessary for Interaction with Its Substrate, the Stimulatory G Protein Gsα. Toxins 2015, 7, 919-935.

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