A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gsα.

Michael Jobling,Lisa Gotow,Zhijie Yang,Randall Holmes

doi:10.3390/toxins7030919

Michael Jobling, Lisa Gotow + Show 2 more

Open Access

https://doi.org/10.3390/toxins7030919

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Abstract

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.

Highlights

Cholera toxin (CT) is a major virulence factor produced by Vibrio cholerae, the causative agent of human cholera [1]
We introduced alanine substitutions into CT holotoxin at selected surface-exposed positions near the CTA1 active site, screened the purified, nicked and reduced holotoxin variants in vitro to identify ones that retained full or nearly-full ability to ADP ribosylate DEABAG but exhibited decreased ability to ADP ribosylate Gsα, and thereby identified specific amino acid residues in CTA1 that likely participate in recognition and binding interactions with Gsα
Fragment in complex with ARF6-GTP and NAD, we selected several surface-exposed residues near the active-site cleft to investigate as possible contributors to the interaction interface between CTA1 and

Summary

Introduction

Cholera toxin (CT) is a major virulence factor produced by Vibrio cholerae, the causative agent of human cholera [1]. Disruption of its interaction with the “active site” loop; and (3) increased flexibility of the “active site” loop enabling it be displaced and permit entry of the substrates NAD and the target arginine residue of Gsα into the active site [10] Additional support for this model was provided by crystal structures of a CT variant with a Y30S substitution in the “activation” loop that permitted the holotoxin to exhibit intrinsic enzymatic activity without any requirement for proteolysis or reduction [9]. We introduced alanine substitutions into CT holotoxin at selected surface-exposed positions near the CTA1 active site, screened the purified, nicked and reduced holotoxin variants in vitro to identify ones that retained full or nearly-full ability to ADP ribosylate DEABAG but exhibited decreased ability to ADP ribosylate Gsα, and thereby identified specific amino acid residues in CTA1 that likely participate in recognition and binding interactions with Gsα

Results and Discussion

Generation of CT Variants by Site Directed Mutagenesis

Protein Production and Purification

Enzymatic Assays

In Vivo Toxicity Assays on Mouse Y1 Adrenal Cells

Conclusions