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.