PDF(Pubmed)
Abstract：
Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA\'s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.
摘要：
蓖麻毒素是一种有效的植物毒素，通过从sarcin-蓖麻毒素环（SRL）中去除腺嘌呤来靶向真核核糖体，rRNA的高度保守的茎环。作为生物恐怖主义的B类药剂,它是用抗体和酶阻断抑制剂进行治疗性干预的主要靶标,因为不存在针对蓖麻毒素的有效疗法。蓖麻毒素A亚基(RTA)通过在远程位点与P-茎蛋白结合来使SRL脱嘌呤。已通过生化方法和X射线晶体学广泛研究了P-茎蛋白对RTA的N-糖苷酶活性的刺激。目前对RTA脱嘌呤机制的理解完全依赖于游离状态并与过渡态类似物络合的酶的X射线结构。迄今为止，我们对RTA活性的构象动力学和变构调节的证据很少，可以在抑制剂的合理设计中加以利用。因此，我们的主要目标是应用溶液NMR技术来探测RTA中残基特定的结构和动态耦合活性，作为理解变构网络功能含义的先决条件。在本报告中，我们介绍了267个残基RTA在游离状态下的从头序列特异性酰胺和侧链甲基化学位移分配，并与代表核糖体P的相同C末端序列的11个残基肽（P11）复合。茎蛋白。这些分配将促进未来的研究，详细说明与抑制剂复合的RTA中结合诱导的构象变化的传播，抗体，和生物学相关的目标。
