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.

II组内含子是经过自我剪接并从信使前RNA中去除的酶。Iheyensis的II型内含子在自拼途径的各个阶段（水解前，水解后和无配体状态）的X射线结构揭示了内含子活性位点中复杂的原子相互作用网络。业已证实，由四个金属离子（K1，K2的K ^+位和M1，M2的Mg ²⁺）组成的异核金属离子簇对于功能至关重要。Ca ²⁺取代Mg ^2+会导致酶活性降低。X射线结构不仅为Mg ²⁺和Ca ²⁺建模提供了可能性结合II组内含子的活性位点并定量估计Mg ²⁺与Ca ²⁺偏好的能量学，还探讨了响应二价金属离子取代的相对结构和动力学差异。因此，使用X射线结构作为模板我们进行分子动力学模拟比较镁之间的结构和动力学的差异²⁺和Ca ²⁺在拼接途径的各个阶段结合的II组内含子的活性位点（即，预水解，后水解和无配体状态）。M1，M2位点的Mg ²⁺与Ca ²⁺选择性的定量估计证实了内含子活性位点相对于Ca的Mg ²⁺偏好性²⁺。相对于Mg 2 ^+， Ca ²⁺在内含子活性位点中的水合程度更高。在Mg ²⁺和Ca ²⁺结合的预水解状态之间，M2周围的局部环境（结合的亲核水，与易磷酸酯的相互作用）显着不同。在水解后状态下，水解产物的外显子部分与M1直接相互作用，而内含子部分在我们的MD轨迹中具有高度的灵活性。M1，M2位的溶剂暴露在水解前状态最少，在无配体状态最高，在水解后中间。