Predicting an intrinsic conformational twist in Card1: an in silico study

Type III CRISPR systems use cA_n second messengers to activate CARF-containing effectors. Card1, a key auxiliary protein, binds cA₄ via its CARF domain, activating its REase domain for ssRNA and ssDNA cleavage. The findings revealed a strong binding interaction between cA₄ and the CARF domain, with each nucleotide forming at least four hydrogen bonds that stabilize the cA₄ binding pocket and restrict its expansion. The conformational change was characterized by a pronounced swinging motion that enlarges the binding pocket, accompanied by a slight twisting motion between the two chains of the CARF domain. Despite significant conformational changes in the CARF domain, the two chains of Card1 exhibited high rigidity, with interchain conformational changes being much smaller than those of the entire complex. The findings further revealed that Card1 undergoes a twist of approximately 12.0° compared to the experimental structure. This study not only provides a theoretical basis for optimizing CRISPR-based genetic tools but also offers important insights into the Type III CRISPR immune mechanism.

All simulations were performed using the AMBER24 package. For the all-atom model, the AMBER ff19SB force field was used. For the coarse-grained model, the SIRAH force field was employed. GaMD simulations were applied using a total potential energy boosting. The large conformational changes were investigated by coarse-grained MD simulations. The CPPTRAJ program was used to calculate structural parameters-including distances, native contacts, RMSD, secondary structure, PCA, angles, and dihedral angles.