Integrative computational simulations and functional assays decode allosteric dysregulation in a pathogenic SIRT6 variant.

SIRT6, a critical member of the NAD-dependent deacetylase family, plays a central role in regulating key biological processes such as DNA repair, transcriptional regulation, aging, and tumor suppression. Although multiple SIRT6 mutations have been identified across cancer types, the structural mechanisms underlying their functional impact remain largely unclear. In this study, we demonstrate that L197P, a point mutation identified in colorectal cancer, significantly impairs the catalytic activity of SIRT6 by destabilizing the substrate-binding loop (B-loop) and reducing substrate binding affinity. Molecular dynamics simulations revealed that the L197P mutation weakens intermolecular interactions between SIRT6 and its acetylated substrate, while also increases the catalytic distance between NAD and the active-site residues. Furthermore, Markov state model analysis indicates that the emergence of a novel inactive conformational state in the mutant, suggesting diminished catalytic efficiency. Consistently, enzymatic assays confirm a six-fold reduction in deacetylation efficiency of the L197P mutant compared with wild-type SIRT6. Collectively, our results elucidate the structural basis by which cancer-associated mutations compromise SIRT6 function, providing mechanistic insights into its allosteric regulation and potential implications for tumorigenesis.