PAZ Domain Pivoting is the Rate-Limiting Step for Target DNA Recognition in the Middle Region of Thermus thermophilus Argonaute

Thermus thermophilus Argonaute (TtAgo) is a DNA-guided programmable endonuclease with emerging applications in genome engineering, yet the rate-determining dynamic mechanisms governing its transition from guide-target hybridization to catalytic activation remain unresolved. Here, we employ molecular dynamics simulations and the Traveling-salesman-based Automated Path Searching (TAPS) approach to dissect the target DNA recognition in the middle region (nt 9–12) of TtAgo. We designed two paths to tackle this problem: one assumed that coordination of the target DNA backbone occurs before base-pairing between the target and guide DNA; the other hypothesized a concerted transition without preferred order between backbone-coordination and base-pairing. While the first path exhibits two high energy barriers (12.45 and 14.12 kcal/mol), the second path is featured by a single rate limiting barrier (12.56 kcal/mol) and therefore more probable to occur. Crucially, the flexible PAZ domain in both scenarios dominates the three rate limiting barrier steps driving bidirectional cavity modulation through pivoting motions. These findings underscore the PAZ domain’s indispensable role in manipulating DNA recognition in the middle region, offering mechanistic insights for engineering high-efficiency Argonaute variants by targeting domain plasticity.