Controlled Trafficking of Multiple and Diverse Cations Prompts Nucleic Acid Hydrolysis

Abstract

Recent in crystallo reaction intermediates have detailed how nucleic acid hydrolysis occurs in the RNA ribonuclease H1 (RNase H1), a fundamental metalloenzyme involved in maintaining the human genome. At odds with the previous characterization, these in crystallo structures unexpectedly captured multiple metal ions (K⁺ and Mg²⁺) transiently bound in the vicinity of the two-metal-ion active site. Using multi-microsecond all-atom molecular dynamics and free-energy simulations, we investigated the functional implications of the dynamic exchange of multiple K⁺ and Mg²⁺ ions at the RNase H1 reaction center. We found that such ions are timely positioned at nonoverlapping locations near the active site, at different stages of catalysis, being crucial for both reactants’ alignment and leaving group departure. We also found that this cation trafficking is tightly regulated by variations of the solution’s ionic strength and is aided by two conserved second-shell residues, E188 and K196, suggesting a mechanism for the cations’ recruitment during catalysis. These results indicate that the controlled trafficking of multication dynamics, opportunely prompted by second-shell residues, is functionally essential to the complex enzymatic machinery of the RNase H1. These findings improve the current knowledge on the catalysis of the RNase H1 andopen new catalytic possibilities for other metalloenzymes including, but not limited to, CRISPR-Cas9, group II intron ribozyme and the human spliceosome.