Structural basis for nicotinamide cleavage andADP-ribose transfer by NADⴙ-dependent Sir2histone兾protein deacetylasesKehao Zhao*†, Robyn Harshaw*†‡, Xiaomei Chai*, and Ronen Marmorstein*§¶*The Wistar Institute,‡Department of Biochemistry and Biophysics, School of Medicine, and§Department of Chemistry, University of Pennsylvania,Philadelphia, PA 19104Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved April 2, 2004 (received for review February 13, 2004)Sir2 enzymes are broadly conserved from bacteria to humans andhave been implicated to play roles in gene silencing, DNA repair,genome stability, longevity, metabolism, and cell physiology.These enzymes bind NADⴙand acetyllysine within protein targetsand generate lysine, 2ⴕ-O-acetyl-ADP-ribose, and nicotinamideproducts. To provide structural insights into the chemistry cata-lyzed by Sir2 proteins we report the high-resolution ternary struc-ture of yeast Hst2 (homologue of Sir two 2) with an acetyllysinehistone H4 peptide and a nonhydrolyzable NADⴙanalogue, carba-NADⴙ, as well as an analogous ternary complex with a reactionintermediate analog formed immediately after nicotinamide hy-drolysis, ADP-ribose. The ternary complex with carba-NADⴙrevealsthat the nicotinamide group makes stabilizing interactions withina binding pocket harboring conserved Sir2 residues. Moreover, anasparagine residue, N116, strictly conserved within Sir2 proteinsand shown to be essential for nicotinamide exchange, is in positionto stabilize the oxocarbenium intermediate that has been pro-posed to proceed the hydrolysis of nicotinamide. A comparison ofthis structure with the ADP-ribose ternary complex and a previ-ously reported ternary complex with the 2ⴕ-O-acetyl-ADP-ribosereaction product reveals that the ribose ring of the cofactor and thehighly conserved␤1–␣2 loop of the protein undergo significantstructural rearrangements to facilitate the ordered NADⴙreactionsof nicotinamide cleavage and ADP-ribose transfer to acetate.Together, these studies provide insights into the chemistry ofNADⴙcleavage and acetylation by Sir2 proteins and have impli-cations for the design of Sir2-specific regulatory molecules.The Sir2 (silent information regulator 2) or sirtuin family ofdeacetylases requires NAD⫹as a cofactor to hydrolyze theacetyl moiety of an acetyllysine within protein targets to regulatediverse biological functions, including gene silencing, genomestability, longevity, metabolism, and cell physiology (for reviews,see refs. 1 and 2). The mechanism for Sir2 activity has beenextensively studied at both structural and enzymatic levels.Structural studies reveal that the Sir2 proteins contain a struc-turally conserved elongated core domain containing a largeRossmann fold at one end, a structurally more variable zinc-binding motif at the opposite end, and a series of loops con-necting these regions and forming a cleft in the central region ofthe core domain (3–6). The acetyllysine and NAD⫹cosubstratesbind to opposite sides of the cleft and highlight the region of thecore domain containing the highest degree of sequence conser-vation within the Sir2 proteins, implying a conserved catalyticmechanism (3, 7). Biochemical and structural studies reveal thatthe deacetylation of acetyllysine is coupled to the hydrolysis ofNAD⫹to nicotinamide and 2⬘-O-acetyl-ADP-ribose (8, 9).A detailed structural understanding of how the Sir2 proteinsmediate nicotinamide cleavage and ADP-ribose transfer to acetatehas been hampered by the difficulty in trapping a Sir2 proteinbound to a form of NAD⫹containing an ordered nicotinamidegroup (6, 7, 10, 11). To visualize the nicotinamide group of NAD⫹bound to a Sir2 protein, we now report the high-resolution crystalstructure of the Saccharomyces cerevisiae Sir2 homologue, yHst2,bound to an acetyllysine-containing histone H4 peptide and car-banicotinamide adenine dinucleotide (carba-NAD⫹). Carba-NAD⫹is an inhibitor of NAD⫹glycohydrolases and ADP-ribosyltransferases (12, 13), where the␤-D-ribonucleotide ring ofthe nicotinamide ribonucleoside moiety of NAD⫹is replaced by a2,3-dihydroxycyclopentane ring, which significantly disfavors hy-drolysis of the pyridinium-N-glycoside bond (14). In this study, wealso report on the structure of a corresponding ternary complexwith ADP-ribose, representing an intermediate mimic formed afternicotinamide cleavage. A comparison of these structures reveals aconserved nicotinamide-binding site and the mechanistic detailsunderlying nicotinamide cleavage from NAD⫹. A further compar-ison of these two structures with a previously reported yHst2ternary complex with the 2⬘-O-acetyl-ADP-ribose reaction productreveals how the NAD⫹reactions of nicotinamide cleavage andADP-ribose transfer to acetate are coordinated through significantconformational changes of the ADP-ribose ring of the NAD⫹cofactor and the␤1–␣2 loop of the protein during catalysis. Theimplications of these studies for the design of compounds for thespecific inhibition or activation of Sir2 proteins are also discussed.MethodsCrystallization and Structure Refinement. The 64-residue C-terminaldeletion construct of yHst2 (residues 1–294) was purified as de-scribed previously (5, 7). Crystals of yHst2兾carba-NAD⫹兾histoneH4 and yHst2兾ADP-ribose兾histone H4 complexes were grown byusing the vapor diffusion method at room temperature and wereobtained by mixing 2␮l of a complex containing 0.24 mM protein,1 mM histone peptide, and 1 mM NAD⫹analog with 2␮lofreservoir solution containing 2.0 M (NH4)2SO4and 100 mM[bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris),pH 5.5兾6.5, and equilibrating over 0.5 ml of reservoir solution.Before data collection, crystals were cryoprotected with a five-stepgradual transfer into reservoir solution supplemented with higherconcentrations of glycerol to a final concentration of 25% (vol兾vol).Crystallographic data for the yHst2兾carba-NAD⫹兾histone H4 andHst2兾ADP-ribose兾histone H4 crystals were collected at the X12Band X25 beamlines at the National Synchrotron Light Source,respectively. All data were processed with HKL 2000 suite (HKLResearch, Charlottesville, VA). The structures of both complexeswere solved by molecular replacement with the programCNS (15),using protein residues 5–293 from the yHst2兾2⬘-O-acetyl-ADP-ribose兾histone H4 structure (PDB ID code 1Q1A) as a searchmodel (7). Structures were