Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD ؉ and acetyllysine within protein targets and generate lysine, 2-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD ؉ analogue, carba-NAD ؉ , as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD ؉ reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved ␤1-␣2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD ؉ reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD ؉ cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules. T he Sir2 (silent information regulator 2) or sirtuin family of deacetylases requires NAD ϩ as a cofactor to hydrolyze the acetyl moiety of an acetyllysine within protein targets to regulate diverse biological functions, including gene silencing, genome stability, longevity, metabolism, and cell physiology (for reviews, see refs. 1 and 2). The mechanism for Sir2 activity has been extensively studied at both structural and enzymatic levels. Structural studies reveal that the Sir2 proteins contain a structurally conserved elongated core domain containing a large Rossmann fold at one end, a structurally more variable zincbinding motif at the opposite end, and a series of loops connecting these regions and forming a cleft in the central region of the core domain (3-6). The acetyllysine and NAD ϩ cosubstrates bind to opposite sides of the cleft and highlight the region of the core domain containing the highest degree of sequence conservation within the Sir2 proteins, implying a conserved catalytic mechanism (3, 7). Biochemical and structural studies reveal that the deacetylation of acetyllysine is coupled to the hydrolysis of NAD ϩ to nicotinamide and 2Ј-O-acetyl-ADP-ribose (8, 9).A detailed structural understanding of how the Sir2 proteins mediate nicotinamide cleavage and ADP-ribose transfer to acetate has been hampered...

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.

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