Structural basis of SIRT2 pre-catalysis NAD+ binding dynamics and mechanism

Abstract

Sirtuins are an evolutionarily conserved family of NAD⁺-dependent deacylases whose catalytic mechanism remains under active investigation. While previous studies have captured sirtuin reaction intermediates using thioacetyl-lysine analogs, here we report six crystal structures of human SIRT2 in complex with native myristoylated peptides and NAD⁺, revealing the sequence of changes from initial NAD⁺ binding to the formation of intermediate I. Our structures provide direct structural evidence for: (1) zinc-binding domain shift during NAD⁺ entry, (2) water-mediated hydrogen-bond formation that disrupts nicotinamide aromaticity preceding cleavage, and (3) the formation of intermediate I. Additionally, we determined the structures of two functionally critical mutants (SIRT2^F96A and SIRT2^H187A), demonstrating their roles in stabilizing NAD⁺ in a productive conformation. These findings complete the comprehensive structural framework for the sirtuin deacylation mechanism and highlight key residues governing catalytic efficiency.