Jobling Michael G, Gotow Lisa F, Yang Zhijie, Holmes Randall K
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.
Toxins (Basel). 2015 Mar 18;7(3):919-35. doi: 10.3390/toxins7030919.
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.
霍乱腹泻的发病机制需要霍乱毒素（CT）介导肠细胞中刺激性G蛋白（Gsα）的腺苷二磷酸（ADP）核糖基化。CT是一种AB5毒素，其无活性的CTA1结构域通过CTA2与五聚体受体结合B亚基相连。与NAD +和GTP结合的ADP核糖基化因子6（ARF6 - GTP）复合的变构激活CTA1片段在构象上与全毒素中的CTA1结构域不同。评估了位于CTA1催化位点附近的一个表面暴露的旋钮、一个短α螺旋（分别由无活性CTA1中的“活性位点”和“激活”环重排形成）和一个ADP核糖基化转环基序（ARTT）在识别Gsα中的可能作用。测试了在这些CTA1基序内表面暴露残基处有一个、两个或三个丙氨酸取代的CT变体组装成全毒素的能力以及对Gsα和二乙氨基 -（苄叉氨基）-胍（DEABAG，一种预计适合CTA1活性位点的小底物）的ADP核糖基化活性。在H55、R67、L71、S78或D109处有单个丙氨酸取代的变体对DEABAG具有几乎野生型的活性，但对Gsα的活性显著降低，这表明天然CTA1中的相应残基参与识别Gsα。由于在这些位置有多个取代的几个变体对Gsα仍保留部分活性，CTA1中的其他残基可能也参与识别Gsα。
