Metal-ion coordination drives active-site remodeling for backsplicing in circularly permuted group II introns

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.