Structure-based thermodynamics of ion selectivity (Mg 2+ versus Ca 2+ and K + versus Na + ) in the active site of the eukaryotic lariat group II intron from algae Pylaiella littoralis .

Group II introns are metalloenzymes that can catalyze self-splicing. Recently, the crystal structures of the eukaryotic group IIB lariat intron from the brown algae Pylaiella littoralis have been reported for two intermediate states (pre-hydrolytic (2s) and post-hydrolytic) along the self-splicing pathway. Three characteristic metal-ion binding sites (M1 and M2 sites for catalytic Mg 2+ ions, and K1 site for K + ) in the catalytic pocket of the lariat intron have been identified and proposed to be crucial for self-splicing. Using the X-ray structures as a template, we quantitatively estimated the energetics of divalent (Mg 2+ versus Ca 2+ ) and monovalent (K + versus Na + ) ion selectivity and established a direct link between the energetics and structures of this lariat intron (bound to cognate and near-cognate metal ions). Molecular dynamics (MD) free energy simulations showed that the lariat intron was strongly selective between divalent metal ions. The strength of divalent metal-ion selectivity was noticeably high in the post-hydrolytic state (ΔΔ G ≈ 20 kcal mol -1 ) relative to its pre-hydrolytic (2s) state (ΔΔ G ≈ 13 kcal mol -1 ). Quantum chemical calculations ensured that the sign of the estimated divalent metal-ion selectivity was correct. The M1-binding pocket was less solvent-exposed in the case of the post-hydrolytic state relative to the pre-hydrolytic (2s) state, which boosted the metal-ion selectivity of the former. Surprisingly, in contrast to the bacterial linear group II intron, the lariat intron was found to be non-selective between monovalent ions (K + versus Na + ). The interaction network in the first coordination shell of Ca 2+ in the M1-binding pocket was different relative to Mg 2+ . Mg 2+ substitution by Ca 2+ resulted in the substitution of a single M1-RNA interaction by the M1-water interaction. In the pre-hydrolytic (2s) state, Ca 2+ substitution completely disrupted the M1⋯5'-exon interaction; thus, the nature of the divalent metal ion is critical for catalysis. The interaction network in the M2 site was independent of the nature of the divalent metal ions (Mg 2+ or Ca 2+ ). The monovalent ion was loosely bound in the wet binding pocket (K1 site) of the lariat intron; thus, the substitution of K + by Na + could not significantly alter the free energy of the complex. The metal ion selectivity was dependent on the solvent accessibility of the metal-ion-binding-pocket, dry pocket enhanced the selectivity.