金属离子配位驱动环状排列 II 组内含子活性位点重塑以完成反向剪接
Metal-ion coordination drives active-site remodeling for backsplicing in circularly permuted group II introns
研究团队解析了来自 Gluconacetobacter diazotrophicus PAL5 的环状排列（CP）II 组内含子 Gdi-1 在分支到反向剪接不同阶段、不同离子条件下的高分辨率冷冻电镜结构。在 K+/Mg2+、K+/Ca2+ 和 Cs+/Mg2+ 条件下的比较显示，有效的反向剪接依赖离子特异的配位网络，这些网络调控 3′ 剪接位点（3′SS）对接和催化中心形成。研究还鉴定出一个此前未被认识的高选择性单价阳离子结合位点 K3，它在结构上将 γ–γ′ 相互作用与异核四金属核心的重组装偶联，从而促进第二步活性位点的形成。
来源：Nucleic Acids Research · doi.org
Circularly permuted (CP) group II introns are self-splicing ribozymes that undergo a sequential branching and backsplicing reaction to yield branched introns and circular exons (circRNAs). While the initial branching step is structurally characterized, the mechanistic principles that govern the subsequent large-scale active-site remodeling and stringent metal-ion specificity required for backsplicing remain poorly understood. Here, we present a series of high-resolution cryo-EM structures of the CP group II intron from Gluconacetobacter diazotrophicus PAL5 (Gdi-1), capturing the ribozyme at discrete stages along the branching -to- backsplicing pathway under distinct ionic conditions. Comparative analyses under K+/Mg2+, K+/Ca2+, and Cs+/Mg2+ conditions demonstrate that productive backsplicing relies on ion-specific coordination networks that govern 3′ splice site (3′SS) docking and catalytic center formation, revealing the precise metal-ion-coordinated conformational rearrangements and dynamic splice-site exchange mechanisms. Crucially, we identify a previously unrecognized, highly selective monovalent cation-binding site, K3, that structurally couples the γ–γ′ interaction to reassembly of the heteronuclear four-metal core, thereby promoting productive second-step active-site formation. Together, these findings establish a structural basis for metal-ion-dependent active-site remodeling during group II intron catalysis and provide mechanistic insights into metal-ion selectivity in circRNA biogenesis.