Principle of K+/Na+ selectivity in the active site of group II intron at various stages of self-splicing pathway

Abstract

Group II introns are ribozymes which can catalyze its own splicing and relegate itself. They share common structural features and are evolutionarily related to eukaryotic spliceosome. Hence, group II introns are an excellent model system for understanding the mechanism of RNA-splicing in gene expression. Recent advancement of structural studies has provided x-ray structures of group II intron at different stages (Pre-hydrolytic, post-hydrolytic and free intron) of splicing pathway and revealed heteronuclear metal ion cluster (two potassium; K1 and K2, two magnesium; M1 and M2) in the active site as common structural feature of group II introns. It is believed that these four metal ions are very crucial for catalysis. K+ is very important for the function and buffers containing only Na+ can destroy the function of group II introns. Despite the available 3D structures of different stages of self-splicing, the energetic origin of K+ selectivity over Na+ is not known. However, these structures provide sufficiently good models for directly computing the energetics of K+ vs Na+ discrimination that is required for understanding self-splicing. Here, we report extensive (∼550 ns) of structure-based molecular dynamics free energy simulations that quantitatively estimated K1 vs Na1 discrimination. We show that the strength of discrimination varies along the self-splicing pathway. The free and pre-hydrolytic states are highly selective for K1 and the selectivity is lost in the post-hydrolytic state. The simulations further reveal that Na1 in the free and pre-hydrolytic state is trapped with an unsatisfied first coordination shell in the active site, which is responsible for large discrimination. The Na1 in the active site of post-hydrolytic state allow water entry and satisfy the Na1 bonding requirement, resulting in low discrimination. The results not only give insights into the experimentally unresolved Na1 bound complexes but also provide the link between structures and computed energetics.