The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near-perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (VHHs) previously reported to recognize epitopes in proximity to RTA's active site. Three of the VHHs, V2A11, V8E6, and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies ("intrabodies"). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTA's active site and interact with key catalytic residues. V2G10, by contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four VHHs also penetrated/occluded RTA's active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.toxin, antibody, structure, intracellular.

中文翻译：

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通过针对活性位点的单域抗体对蓖麻毒素进行细胞内中和。

植物毒素蓖麻毒素的极强效力归因于其酶亚基 RTA，它能以近乎完美的效率灭活哺乳动物核糖体。在此，我们在功能和结构水平上对七种羊驼单域抗体 (VHH) 进行了表征，这些羊驼单域抗体 (VHH) 先前报道可识别 RTA 活性位点附近的表位。其中三种 VHH（V2A11、V8E6 和 V2G10）在体外是有效的 RTA 抑制剂，当表达为细胞内抗体（“胞内抗体”）时，可以保护 Vero 细胞免受蓖麻毒素的侵害。晶体结构分析表明，V2A11 和 V8E6 的互补决定区 3 (CDR3) 元件穿透 RTA 的活性位点并与关键催化残基相互作用。相比之下，V2G10 位于酶袋顶部并阻碍底物可及性。其他四个 VHH 也穿透/封闭了 RTA 的活性位点，但缺乏足够的结合亲和力来竞争 RTA-核糖体相互作用。高亲和力、单域抗体的细胞内递送可能为开发针对蓖麻毒素的对策提供新途径。毒素、抗体、结构、细胞内。