title: A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gsα.
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.
authors: Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver, 12800 E 19th Ave, Aurora, CO 80045, USA. michael.jobling@ucdenver.edu.
authors: Michael G
authors: MG
authors: Jobling
authors: Department of Biology, Metropolitan State University of Denver, P.O. Box 173362, CB 53, Denver, CO 80217, USA. lgotow@msudenver.edu.
authors: Lisa F
authors: LF
authors: Gotow
authors: Atila Biosystems Inc., 740 Sierra Vista Ave, Unit E, Mountain View, CA 94043, USA. zhijie.yang@atilabiosystems.com.
authors: Zhijie
authors: Z
authors: Yang
authors: Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver, 12800 E 19th Ave, Aurora, CO 80045, USA. randall.holmes@ucdenver.edu.
authors: Randall K
authors: RK
authors: Holmes
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D014644:Genetic Variation; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002772:Cholera Toxin; D019205:GTP-Binding Protein alpha Subunits, Gs; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse