Mechanistic basis of N-terminal domain-mediated allostery in SIRT6: integrating molecular dynamics simulations and biochemical assays.

SIRT6, a pivotal member of the NAD⁺-dependent deacetylase superfamily, regulates critical biological processes, including DNA repair, transcriptional regulation, and aging. The deacetylase activity of SIRT6 is allosterically coupled to NAD⁺ binding, enabling site-specific removal of acetyl moieties from lysine substrates. Despite its physiological significance, the structural mechanisms underlying the allosteric regulation mediated by its N-terminal domain (NTD) have remained elusive. In this study, we establish that the NTD of SIRT6 plays an indispensable role in preserving the catalytic geometry by maintaining the NAD⁺ pocket conformation and stabilizing substrate coordination. Molecular dynamics simulations revealed that truncation of the NTD induces an open-state NAD⁺ pocket configuration, accompanied by a reduction in NAD⁺ binding affinity and an increase in the catalytic distance between NAD⁺ and the acetylated lysine substrate. Consistently, enzymatic assays demonstrated a twofold decrease in deacetylation efficiency in NTD-truncated enzyme compared to wild-type SIRT6. These results provide novel mechanistic insights into the NTD-mediated allosteric network essential for SIRT6 catalysis, offering a structural framework for developing modulators targeting this regulatory domain.