Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Abstract

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...

Chemistry of acetyl transfer by histone modifying enzymes: structure, mechanism and implications for effector design

“…Instead, the carbonyl of a conserved asparagine residue, shown to be essential for nicotinamide hydrolysis, is in position to stabilize a positively charged oxocarbenium ion. Also consistent with this mechanism is a structure of a ternary complex with yHst2, acetyl-lysine and the intermediate analogue, ADP-ribose that reveals that the nicotinamide ribose ring rotates about 901 relative to its orientation in carba-NAD þ and now places carbonyl oxygen of acetyl lysine in position to carry out nucleophilic attach on the 1 0 carbon on the a-face of the nicotinamide ribose ring (Zhao et al, 2004). These differing models for the mode of nicotinamide hydrolysis also have implications for the mode of nicotinamide inhibition.…”

“…This mechanism necessitates formation of an oxocarbenium ion intermediate after hydrolysis of the nicotinamide and before nucleophilic attack of the acetyl group of acetyl-lysine (Figure 4b). Consistent with this mechanism is a crystal structure of a ternary complex of yHst2 with acetyl-lysine and a non-hydrolysable analogue of NAD þ , carba-NAD þ , in which the in-ring oxygen of the nicotinamide ribose is replaced with a carbon atom (Zhao et al, 2004). This structure shows that the acetyl group of acetyl-lysine is hydrogen bonded to the 2 0 -and 3 0 -OH groups of the nicotinamide ribose and is not in position to carry out nucleophilic attack at the 1 0 carbon of the nicotinamide ribose ring.…”

“…There is one reported structure of a full-length sirtuin protein, yeast Hst2, and this structure shows a homotrimeric structure that appears to be in an autoinhibited form. In particular, this structure shows that residues N-terminal to the catalytic domain sit within the acetyllysine binding pocket of another protein subunit of the trimer and that a C-terminal helix sits within the NAD þ binding site of the same subunit (Zhao et al, 2004). To what extent this feature is conserved within other sirtuins is unknown; however, it appears unlikely since regions N-and C-terminal to the conserved catalytic core show no sequence conservation among the sirtuin proteins.…”

“…A second model argues that the inhibitory nicotinamide molecule binds to a nearby 'D-pocket' that is distinct from the C-pocket (Figure 4c). This model was initially based on a structure of yHst2 in complex with lysine-16 histone H4 and a reaction intermediate analogue, ADP-ribose, that modeled an incoming nicotinamide molecule within the D-pocket (Zhao et al, 2004). More recently, a crystal structure of nicotinamide within the D-pocket was obtained by socking nicotinamide into crystals containing yHst2, lysine-16 histone H4 and reaction intermediate analogue designed to mimic the oxocarbenium ion, ADP-HPD (Sanders et al, 2007).…”

Structural basis for allosteric stimulation of Sir2 activity by Sir4 binding

“…The structures of sirtuins with substrate peptides reveal that the substrate peptides are bound at the entry of the cleft primarily via hydrogen bonds between main chain atoms of the enzyme and the substrate peptides (Avalos et al 2002;Zhao et al 2003b). The acetyl-lysine of the substrate peptides is inserted into a hydrophobic channel, and, as predicted, the nicotinamide moiety of NAD is forced into the C pocket, where the space is more restricted (Avalos et al 2004;Zhao et al 2004). Based on this observation, a ground-state destabilization model of NAD-dependent protein lysine deacetylation has been proposed (Avalos et al 2004).…”