Department of Chemistry, Frick Laboratory , Princeton University , Princeton , New Jersey 08544 , United States.
Department of Biochemistry , Albert Einstein College of Medicine , Bronx , New York 10461 , United States.
J Am Chem Soc. 2018 Sep 19;140(37):11791-11799. doi: 10.1021/jacs.8b07334. Epub 2018 Sep 10.
Split inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the intein pair are ligated together in a traceless manner. Recently, a family of naturally split inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split intein complementation in which localized structural rearrangements are used to drive folding and regulate protein-splicing activity.
分裂内含肽通过转译调控发生蛋白剪接，这是一种将融合到内含肽对的蛋白序列进行无痕迹连接的翻译后修饰。最近，人们鉴定了一组天然分裂内含肽，它们在一级序列的非典型位置发生分裂。这些非典型分裂内含肽在蛋白质工程应用中具有很大的应用潜力；然而，由于独特的拓扑限制，它们的关联机制尚不清楚，而且必须与之前表征的典型分裂内含肽不同。在这里，我们使用共识设计策略生成一个具有大大提高的活性且易于进行详细生化和生物物理分析的非典型分裂内含肽对 (Cat)。受 Cat 的溶液结构的指导，我们表明，片段的关联涉及由疏水相互作用驱动的无序到有序的结构转变。这种分子识别机制满足内含肽折叠的拓扑限制，并且重要的是，确保在片段互补之前不会发生过早的化学变化。我们的数据为分裂内含肽互补提供了一个共同的蓝图，其中局部结构重排用于驱动折叠并调节蛋白剪接活性。
