Mg²⁺ vs Ca²⁺ bound active site of group II intron- A MD study

Group II introns are enzymes which undergo self-splicing and remove itself from pre-messenger RNA. X-ray structures of group II intron of Oceanobacillus iheyensis at various stages of the self-splicing pathway (pre-hydrolytic, post-hydrolytic, and ligand-free state) revealed intricate atomic interaction network in the active site of the intron. It has been confirmed that a heteronuclear metal ion cluster consisting of four metal ions (K1, K2 sites with K⁺ and M1, M2 sites with Mg²⁺) are crucial for function. Substitution of Mg²⁺ by Ca²⁺ results in loss of enzymatic activity. The X-ray structures not only opens up the possibility of modelling Mg²⁺ and Ca²⁺ bound active site of group II intron and quantitatively estimate the energetics of Mg²⁺ vs Ca²⁺ preference but also explore the relative structural and dynamical differences in response to divalent metal ion substitution. Thus, using X-ray structures as a template we performed molecular dynamics simulations to compare structural and dynamical differences between Mg²⁺ and Ca²⁺ bound active site of group II intron at various stages of the splicing pathway (i.e., pre-hydrolytic, post-hydrolytic, and ligand-free state). Quantitative estimation of Mg²⁺ vs Ca²⁺ selectivity at the M1, M2 sites confirmed Mg²⁺ preference at intron active sites relative to Ca²⁺. Ca²⁺ is relatively more hydrated in the intron active site relative to Mg²⁺. The local environment (bound nucleophilic water, interaction with scissile phosphate) around M2 is strikingly different between Mg²⁺ and Ca²⁺ bound pre-hydrolytic state. In the post-hydrolytic state, the exon part of the hydrolysis product is involved in direct interaction with the M1, whereas the intron part is highly flexible in our MD trajectories. Solvent exposure of M1, M2 sites are least in the pre-hydrolytic state, highest in the ligand-free state, and intermediate in the post-hydrolytic state.