Abstract
The CRISPR-Cas9 system’s gene-editing fidelity relies on the HNH nuclease domain’s ability to hydrolyze DNA via metal-ion catalysis. While IscB-ωRNA variants offer advantages for viral delivery due to their small size, their editing efficiency is much lower than that of Cas9. The understanding of how HNH-domain dynamics and metal-ion selection jointly control catalysis is essential for its rational improvement. Here, we employ molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) simulations, and free energy perturbations (FEP) to study the conformation dynamics of the IscB–ωRNA–target DNA complex and cation binding energy in the core HNH(IscB)–DNA region. Our MD simulations revealed that the IscB–ωRNA–DNA complex is overall stable. However, the core HNH(IscB)–DNA region shifts to a larger separation, consistent with recent findings of coupling between HNH motion and activation in IscB–ωRNA–DNA. However, the HNH domain motion may be decreased by Cas9-like H246D/H270N mutations. QM/MM and FEP studies found that three Cas9 activating ions, Mg2+, Mn2+, and Co2+, also have higher binding energy in the mutated HNH(IscB)–DNA region, suggesting that the catalytic efficiencies of IscB–ωRNA could be subjected to the balance between stabilizing ion binding and domain motions.