Untangling the Molecular Mechanism of SpCas9 HNH Domain Checkpoint Activation: A Gear-and-Wedge Fitting Model

Abstract The CRISPR-associated endonuclease Streptococcus pyogenes Cas9 (SpCas9) facilitates site-specific DNA cleavage, with structural transition from the checkpoint state to the catalytic conformation representing a critical activation step. This transition involves an approximately 38 Å displacement of the HNH domain catalytic residue H840 toward the target DNA. However, the molecular determinants governing this large-scale rearrangement of the HNH domain during checkpoint activation, and the conformational routes by which HNH repositioning may occur, remain incompletely understood. To address this knowledge gap, we combined nudged elastic band-guided sampling with unbiased molecular dynamics simulations, reaching a cumulative timescale of 160 μs, and constructed a kinetic model describing HNH domain dynamics during checkpoint activation with Markov state modeling. The mechanistic features suggested by these simulations were further evaluated through biochemical DNA cleavage assays and an intracellular fluorescence reporter system. Our results identify conformational plasticity of the L2 linker and coupled motion of the REC2 domain as key regulatory elements of the HNH domain relocation during SpCas9 checkpoint activation. The model suggests that L2 linker remodels toward a disassembled state to enable release and repositioning of the HNH domain, while the REC2 domain outward displacement guides HNH domain relocation through transient interactions that stabilize activation-associated intermediates. Site-directed mutagenesis of key residues in the L2 linker and REC2 domain significantly reduced DNA cleavage efficiency in both biochemical and cellular assays, supporting their functional importance in SpCas9 activation. Taken together, this study provides an experimentally grounded mechanistic framework for understanding how linker dynamics and REC2 domain coupling regulate HNH domain checkpoint activation, and offers insights for rational engineering of SpCas9 variants with altered activity and specificity.