Abstract:
Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.
摘要:
蓖麻毒素是已知的毒性最强的物质之一,是一种B型生物试剂。由大肠杆菌(STEC)和痢疾志贺氏菌产生的志贺毒素(Stxs)是食源性病原体。没有针对蓖麻毒素或STEC的有效疗法,并且迫切需要抑制剂。蓖麻毒素A亚基(RTA)和Stx2a的A1亚基(Stx2A1)与核糖体P-茎蛋白的C末端结构域(CTD)结合,以对sarcin/蓖麻毒素环进行纯化。尚未探索调节毒素-核糖体相互作用作为抑制策略。因此,检测毒素-核糖体相互作用抑制剂的检测方法的开发仍然是一个关键的需求。在这里,我们描述了一种基于荧光各向异性(FA)的竞争性结合测定,使用衍生自P茎CTD的BODIPY-TMR标记的11聚体肽(P11)来测量3-11个氨基酸的肽对RTA和Stx2A1的P茎袋的结合亲和力。与表面等离子体共振(SPR)测定的亲和力比较表明,尽管两种方法的等级顺序相同,FA测定可以更好地区分通过SPR显示非特异性相互作用的肽。FA测定仅检测与标记的P11竞争的相互作用,并且可以验证抑制剂特异性和作用机制。