Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis

Group II introns require numerous divalent metal ions for folding and catalysis. However, because little information about individual metal ions exists, elucidating their ligands, functional roles and relationships to each other remains challenging. Here we provide evidence that an essential motif at the catalytic center of the group II intron, the AGC triad within domain 5 (D5), provides a ligand for a crucial metal ion. Sulfur substitution of the pro-S_p oxygen of the adenosine strongly disrupts D5 binding to a substrate consisting of an exon and domains 1-3 of the intron (exD123). Cd²⁺ rescues this effect by enabling the sulfur-modified D5 to bind to exD123 with wild type affinity and catalyze 5′ splice site cleavage. This switch in metal specificity implies that a metal ion interacts with D5 to mediate packing interactions with D123. This new D5 metal ion rescues the disruption of D5 binding and catalysis with a thermodynamic signature different from that of the metal ion that stabilizes the leaving group during the first step of splicing, suggesting the existence of two distinct metal ions.