Humberg Christoph, Yilmaz Zahide, Fitzian Katharina, Dörner Wolfgang, Kümmel Daniel, Mootz Henning D
Institute of Biochemistry, University of Münster, Corrensstraße 36, 48149, Münster, Germany.
Nat Commun. 2025 Mar 19;16(1):2723. doi: 10.1038/s41467-025-57596-x.
Split inteins catalyze protein trans-splicing by ligating their extein sequences while undergoing self-excision, enabling diverse protein modification applications. However, many purified split intein precursors exhibit partial or no splicing activity for unknown reasons. The Aes123 PolB1 intein, a representative of the rare cysteine-less split inteins, is of particular interest due to its resistance to oxidative conditions and orthogonality to thiol chemistries. In this work, we identify β-sheet-dominated aggregation of its N-terminal intein fragment as the origin of its low (~30%) splicing efficiency. Using computational, biochemical, and biophysical analyses, we characterize the fully active monomeric fraction and pinpoint aggregation-prone regions. Supported by a crystal structure, we design stably monomeric mutants with nearly complete splicing activity. The optimized CLm intein (Cysteine-Less and monomeric) retains the wild-type's ultra-fast reaction rate and serves as an efficient, thiol-independent protein modification tool. We find that other benchmark split inteins show similar precursor aggregation, suggesting that this general phenomenon arises from the intrinsic challenge to maintain the precursor in a partially disordered state while promoting stable folding upon fragment association.
分裂内含肽通过在自我切割的同时连接其外显肽序列来催化蛋白质反式剪接，从而实现多种蛋白质修饰应用。然而，许多纯化的分裂内含肽前体由于未知原因表现出部分或无剪接活性。Aes123 PolB1内含肽是罕见的无半胱氨酸分裂内含肽的代表，因其对氧化条件具有抗性且与硫醇化学正交而备受关注。在这项工作中，我们确定其N端内含肽片段以β-折叠为主的聚集是其低剪接效率（约30%）的根源。通过计算、生化和生物物理分析，我们表征了完全活性的单体部分并确定了易于聚集的区域。在晶体结构的支持下，我们设计了具有几乎完全剪接活性的稳定单体突变体。优化后的CLm内含肽（无半胱氨酸且为单体）保留了野生型的超快反应速率，并作为一种高效、不依赖硫醇的蛋白质修饰工具。我们发现其他基准分裂内含肽也表现出类似的前体聚集现象，这表明这种普遍现象源于在促进片段缔合时稳定折叠的同时将前体维持在部分无序状态的内在挑战。
