How Do DICER1 Syndrome Mutations Disrupt Catalysis? Unveiling Dicer Metal Binding Architecture and Mechanism of Action Using MD Simulations and QM/MM Calculations.
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The RNA interference (RNAi) pathway regulates gene expression and viral defense and has been harnessed in therapeutic solutions to inhibit otherwise undruggable proteins by preventing translation. Dicer initiates RNAi by generating cleaved RNA products that bind to a target mRNA to promote gene silencing. Mutations to the Dicer catalytic domain cause DICER1 syndrome, which increases the risk of cancers, including early childhood variants. However, the catalytic mechanism remains poorly defined due to the lack of structural data for Dicer bound to a substrate (or substrate mimic) in the presence of divalent ions known to be critical for nuclease activity. This study uses molecular dynamics (MD) simulations to uncover the first atomic level structure of the wild-type Dicer-RNA complex, including the binding pattern of two catalytically essential Mg2+ ions. Subsequently, quantum mechanics/molecular mechanics (QM/MM) techniques are used to elucidate the Dicer catalytic mechanism for phosphodiester bond cleavage. Among three fully characterized pathways, our data suggest catalysis is only feasible when both active site Mg2+ ions are directly coordinated to the RNA substrate and a hydroxide ion nucleophile is bound to an active site Mg2+ ion. Phosphodiester bond hydrolysis proceeds through a two-step mechanism involving a phosphorane intermediate that is stabilized by direct Mg2+-substrate coordination and a hydrogen bond with K1806. This newly identified mechanism is consistent with experimental kinetic data, the impact of mutating the corresponding lysine in mouse Dicer, and the active site architectures and proposed mechanisms for other related nucleases. Directed by the enhanced understanding of wild-type Dicer function, MD simulations subsequently show that six known DICER1 syndrome-causing mutants likely impede catalysis by inducing unique active site disruptions that inhibit Mg2+-ion coordination to the substrate. By furthering our knowledge of the structure and catalytic mechanism of wild-type and mutant Dicer, this work unveils the molecular underpinnings of DICER1 syndrome and opens the door for the development of enhanced RNAi-based therapeutics and biotechnologies.