Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes.

Keywords: EndoU; RNA; allostery; calcium; ribonuclease.

核糖核酸酶（RNases）是一种普遍存在的酶，负责处理或降解RNA，对于细胞功能和免疫反应至关重要。EndoU-like超家族包括在细菌、真核生物以及某些病毒中保守的内切核糖核酸酶，并且与核糖核酸酶A样超家族有古老的进化联系。无论是细菌中的EndoU还是动物中的RNase A都具有相似的折叠结构，且其功能独立于辅因子。相比之下，真核生物中的EndoU催化域需要二价金属离子进行催化，这可能是由于靠近催化核心的N端延伸所致。在此研究中，我们使用了生物物理和计算技术以及in vitro实验来探究人类EndoU的钙依赖性激活。我们确定了与钙结合的EndoU晶体结构，并发现远离催化三联体的钙结合触发了水介导的分子内信号传导及结构变化，通过别构效应激活该酶。钙结合涉及到催化核心和N端延伸中的残基，表明N端延伸与催化核心相互作用以响应钙调节活性。我们的研究结果表明，在所有真核生物EndoU中可能存在类似的机制，突显了一种独特的进化适应性，它将内切核糖核酸酶的活性连接到真核细胞的信号传导上。

关键词： EndoU; RNA; 别构效应; 钙; 核糖核酸酶。