Conformational Transition of the CARF Domain Driven by Binding Free Energy

Type III CRISPR systems provide adaptive immunity against invasion of foreign nucleic acids by generating cyclic oligoadenylate (cAn) second messengers, which activate effector proteins containing CRISPR-associated Rossmann fold (CARF) domains. The apo form of CARF adopts a closed state, distinct from its cA₄-bound open state conformation. To investigate the conformational transition, we performed multiple type molecular dynamics (MD) simulations, revealing a unidirectional conformational shift toward the closed state. This transition was hindered by reduced flexibility in cA₄-binding residues. Notably, the conformational change primarily occurs between the two monomers, with minimal structural rearrangement within individual monomers. Comparative analysis showed that while the number of hydrogen bonds and contacts between CARF and cA₄ decreases in the closed state, intermonomer interactions are strengthened. Binding free-energy calculations between the two chains of CARF further confirmed higher affinity in the closed state. Our findings support an energy-driven conformational change model, providing insights for optimizing CRISPR-based genetic manipulation tools.