Molecular dynamics simulation and QM/MM study of IscB-ωRNA complex

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.