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Oncogene (2007) 26, 5528–5540 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Chemistry of acetyl transfer by modifying : structure, mechanism and implications for effector design

SC Hodawadekar and R Marmorstein

The Wistar Institute and The Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA

The post-translational modification of plays an remodeling that mobilize the histone proteins important role in regulation, a process that within chromatin (Varga-Weisz and Becker, 2006); insures the fidelity of and other DNA histone chaperone proteins that assemble, disassemble transactions. Of the enzymes that mediate post-transla- or replace variant histones within chromatin (Loyola tion modification, the histone acetyltransferase (HAT) and Almouzni, 2004); and post-translational modifica- and (HDAC) proteins that add and tion enzymes that add or remove functional groups to or remove acetyl groups to and from target residues from the histone proteins (Nightingale et al., 2006). within histones, respectively, have been the most exten- The post-translational modifications of histones sively studied at both the functional and structural levels. involve the addition or removal of acetyl, methyl or Not surprisingly, the aberrant activity of several of these groups as well as the reversible transfer of the enzymes have been implicated in human diseases such as and sumo proteins. Of the enzymes that carry cancer and metabolic disorders, thus making them out post-translational modifications of histones, the important drug targets. Significant mechanistic insights proteins that mediate acetyl-transfer onto lysine side into the function of HATs and HDACs have come from chains within the N-terminal histone tails are the most the X-ray crystal structures of these enzymes both alone well-studied at both the functional and structural levels and in liganded complexes, along with associated enzy- (Marmorstein and Roth, 2001a; Marmorstein, 2001b). matic and biochemical studies. In this review, we will The of histones is carried out by a group of discuss what we have learned from the structures and enzymes known as histone acetyl (HATs) related biochemistry of HATs and HDACs and the and histone deacetylases (HDACs) catalyse the reverse implications of these findings for the design of reaction. effectors to regulate gene expression and treat disease. The opposing activity of HATs and HDACs tightly Oncogene (2007) 26, 5528–5540; doi:10.1038/sj.onc.1210619 regulate gene expression and are roughly correlated with gene activation and repression/silencing, respectively Keywords: histone acetyltransferases (HAT); histone deace- (Brownell and Allis, 1996; Mizzen and Allis, 1998). tylases (HDAC); post-translational histone modifications Considering the important role that HATs and HDACs play in gene regulation, it has not come as a surprise that alterations in HAT and HDAC activity have been correlated with human diseases. For example, chromo- somal translocations involving the monocytic leukemia zinc finger protein (MOZ) and p300/CBP HATs are Introduction associated with acute myeloid leukemias and p300/CBP have properties of tumor suppressor proteins (Muraoka DNA within the eucaryotic nucleus is compacted et al., 1996; Giles et al., 1998; Chaffanet et al., 2000; through its association with two copies each of the Timmermann, 2001). In addition, class I/II HDACs highly basic histone proteins H2A, H2B, H3 and H4 to have emerged as attractive targets for cancer therapy, as form core particles (Luger et al., 1997) that HDAC inhibitors have been found to have potent assemble into a dynamic structure known as chromatin and specific anticancer activities in preclinical studies (Woodcock, 2006). The appropriate regulation of (Kelly et al., 2002, 2003; Marks and Jiang, 2005; Bolden chromatin orchestrates several nuclear processes such et al., 2006). Finally, the class III HDACs, or , as DNA , replication, repair, mitosis and have been implicated in diverse biological processes apoptosis. The proteins, or associated protein com- including physiology, and longevity plexes, that regulate chromatin activity fall into at least (Haigis and Guarente, 2006). Consistent with this three different groups; ATP-dependant chromatin regulation, activators related to the polyphenol have been shown to suppress age-associated diseases such as type II diabetes, obesity and cancer Correspondence: Dr R Marmorstein, Department of Chemistry, The Wistar Institute, University of Pennsylvania, 3601 Spruce Street, (Wood et al., 2004a, b; Kobayashi et al., 2005; Parker Room 327, Philadelphia, PA 19104, USA. et al., 2005; Anekonda and Reddy, 2006; Baur et al., E-mail: [email protected] 2006). Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5529 The pivotal transcriptional role played by the enzymes Structure of nuclear HATs that catalyse acetyl-transfer, and their association with human diseases as described above has motivated The structure of HAT domains from several members of detailed structural and mechanistic studies on their the Gcn5/PCAF family has been determined by X-ray mode of action. In this review, we will summarize what crystallography and nuclear magnetic resonance, both has been learned from the structure of HATs alone and in complex with substrates (Clements et al., and HDACs together with the associated biochemistry 1999; Lin et al., 1999; Rojas et al., 1999; Trievel et al., with a particular focus on the implications of these 1999; Yan et al., 2000, 2002; Poux et al., 2002; Poux and studies for understanding catalytic mechanism and for Marmorstein, 2003). These structures reveal that the A, designing small molecule effectors that target these B and D regions of the GNAT proteins form a central enzymes for the treatment of human diseases such as core that is structurally homologous to other GNAT cancers. proteins and mediates conserved Ac-CoA interactions (Figure 1a). The Gcn5/PCAF HAT domains also contain flanking N- and C-terminal domains that show structural divergence to other GNAT proteins. Together, the core and N- and C-terminal flanking Overview of nuclear HATs regions of the Gcn5/PCAF HAT domain form a pronounced cleft over the core region and flanked by Nuclear or A-type HAT proteins, to distinguish them the N- and C-terminal regions that forms the binding from the cytoplasmic B-type HATs, can be grouped into site for the acetyl-lysine-bearing histone substrate at least three families, Gcn5/PCAF (for its founding (Figure 1a). Aside from acetyl-lysine contacts that are member yeast Gcn5 and its ortholog in human, PCAF), mediated by the HAT core region, nearly all of the MYST (for the founding members MOZ, Ybf2/Sas3, other histone contacts are mediated by the flanking Sas2 and Tip60) and p300/CBP (for the two human N- and C-terminal regions of the Gcn5/PCAF paralogs p300 and CBP) (Sterner and Berger, 2000b; HAT domain implicating these regions for substrate Marmorstein, 2001a). Although other nuclear HAT specificity. families have been identified, such as the steroid receptor The X-ray crystal structure of the Esa1 (Yan et al., coactivators (ACTR/AIB1, SRC1), TAF250, ATF-2, 2000) member of the MYST HAT family bound to CoA their HAT activities have not been studied in detail, so reveals a structurally superimposable core domain and they will not be discussed here. Yeast HAT1 was the first CoA interactions with Gcn5/PCAF, despite the very identified B-type HAT and it is discussed in another limited sequence conservation between these HATs article in this issue of Oncogene (Parthun, 2007). An (Figure 1b). In contrast, the N- and C-terminal regions unusual feature of the nuclear HAT subfamilies is that of the Esa1 HAT domain are structurally divergent from although they employ a common acetyl (Ac)-CoA the corresponding regions of the Gcn5/PCAF HAT and acetyl-lysine substrate and generate the domain. Since the flanking N- and C-terminal regions of same reaction products, CoA and deacetylated lysine, Gcn5/PCAF play an important role in substrate they show significant sequence divergence that is specificity, it is interesting to speculate that this believed to be correlated with their distinct substrate divergent region of Esa1 plays a similar role, although specificities and biological activities. Gcn5 and PCAF there is no direct evidence for this. belong to the GNAT family (Gcn5-related N-acetyl- Although a structure of the HAT domain of p300/ ) that contains three conserved sequence CBP has not yet been reported, biochemical information motifs (A, B and D) that are shared with other indicates that it is structurally distinct from the Gcn5/ acetyltransferases, including serotonin acetyltransferase PCAF and MYST HATs. In particular, it was reported and spermidine acetyltransferase (Neuwald and Lands- that the p300 HAT domain contains two protease- man, 1997). Some of the other GNAT members contain resistant domains connected by a long protease-sensitive another conserved C motif. The MYST proteins contain loop (Thompson et al., 2004). Moreover, the authors sequence homology to the GNAT proteins only in motif demonstrate that this loop is autoinhibitory in its A, and the p300/CBP family has no detectable sequence hypoacetylated form, but undergoes autoacetylation conservation with either the Gcn5/PCAF or MYST for HAT activation suggesting some allosteric regula- HATs. Several HATs within the Gcn5/PCAF and tion of p300 HAT activation similar to how protein MYST family have been shown to exist in multisubunit are activated by of their complexes in vivo and at least one activity of these activation loops. Based on the structural comparisons associated HAT subunits appears to be to target the between the Gcn5/PCAF and MYST HATs, we HATs to , as the recombinant Gcn5 and speculate that the p300/CBP HATs may have a Esa1 HAT proteins strongly prefers free histones structurally conserved central core but divergent N- over nucleosomes (Sterner and Berger, 2000a; and C-terminal domains with additional divergence Marmorstein and Roth, 2001b). Some HATs such as within its activation loop. Taken together, the nuclear PCAF and p300/CBP have also been shown to target HATs appear to contain a conserved core for Ac-CoA non-histone transcription factors such as and binding but otherwise have different components that MyoD for acetylation (Glozak et al., 2005; Zhang and fold around this core to facilitate substrate-specific Dent, 2005). activities.

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5530

Figure 1 Overall structure of HATs. (a) Structure of the Gcn5/PCAF family HAT domain. The structurally conserved core region of Tetrahymena Gcn5 (PDB ID: 1QSN) containing the A and D segments of the GNAT proteins is colored in dark blue with the flanking N- and C-terminal segments colored in aqua. The hydrolysed CoA (red) cofactor and the histone H3 peptide (yellow) substrate are shown in stick figure representation. (b) Structure of the MYST family HAT domain. Yeast Esa1 (PDB ID: 1MJA) bound to Ac-CoA. (red) is color-coded and shown in the same orientation as in (a). (c) of HATs. Active site residues for the yeast Gcn5 (blue) (PDB ID: 1YGH) and yeast Esa1 (PDB ID: 1MJA) (green) members of the Gcn5/PCAF and MYST HATs, respectively, are shown as stick representation on a schematic of the superimposed structures. (d) Catalytic mechanism of HATs. Ternary complex and ping-pong catalytic mechanisms as proposed for the Gcn5/PCAF and MYST HATs, as established from work on the yeast Gcn5 and yeast Esa1 HAT domains, respectively, are illustrated. Ac-CoA, acetyl-coenzyme-A; GNAT, Gcn5-related N-acetyltransferase; HAT, histone acetyltransferase.

Catalytic mechanism of nuclear HATs in vivo studies, revealed that a glutamate residue (E338 in yeast Esa1) that showed structural alignment with the The structure of the nascent yeast Gcn5 HAT domain, corresponding catalytic glutamate residue of Gcn5/ together with mutational and enzymatic studies, reveals PCAF played a similar catalytic role. Based on this that the Gcn5/PCAF HAT enzymes use a ternary observation, it was hypothesized that the Gcn5/PCAF complex mechanism (Tanner et al., 1999; Trievel et al., and MYST proteins employ similar catalytic mechan- 1999) involving deprotonation of the N-e-nitrogen of the isms for catalysis. Subsequent structural, biochemical, target lysine by a conserved glutamate residue (E173 in mutational and enzymatic studies revealed that the yeast Gcn5) within the core domain followed by direct recombinant yeast Esa1 HAT domain employed an nucleophilic attack of the deprotonated nitrogen on the active site (C304 in yeast Esa1) residue to of the Ac-CoA bound cofactor (Figures 1c mediate a ping-pong catalytic mechanism (Yan et al., and d). Initial structural studies on the yeast Esa1 HAT 2002), involving an acetyl-cysteine intermediate (Figures domain, together with mutational and in vitro and 1c and d). This conclusion was supported by the ability

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5531 to trap a crystal structure of a C304 acetylated Esa1 Structural basis for HAT inactivation intermediate, to biochemically use this intermediate to transfer the acetyl group to target lysine and to The only direct structural insights into small molecule- demonstrate that both the E338Q and C304A or mediated inhibition of HAT proteins has come from a C304S showed background levels of HAT activity. crystal structure of the Tetrahymena Gcn5 (tGcn5) HAT Interestingly, a very recent report (Berndsen et al., 2007) domain bound to a modified H3-CoA-20 inhibitor presents kinetic data that are consistent with a four- (Poux et al., 2002) described above (Figure 2a). The subunit piccolo NuA4 complex, containing the Esa1 bisubstrate inhibitor used in this study was prepared catalytic HAT subunit, proceeding through a ternary with an isopropionyl bridge between CoA and peptide complex mechanism involving E338. The authors to more closely mimic the Ac-CoA-lysine intermediate further show that a C304A and C304S mutations in (Lau et al., 2000b). With an IC50 of 300 nM for tGCN5, Esa1 decreased the kcat by only about 2- and 10-fold, this is the most potent inhibitor to the Gcn5/PCAF respectively, leading the authors to conclude that the family of HATs identified to date. A superposition of Esa1 containing piccolo NuA4 complex proceeds the inhibited complex with the ternary tGcn5/CoA/H3 through a ternary complex mechanism that does not peptide complex shows that although the CoA and involve a C304 intermediate. These new results imply an lysine substrate superimpose well, the rest of the peptide apparent discrepancy that will require further analysis, residues go in different directions and all but four of the although it is possible that within different contexts residues that flank the lysine substrate are disordered in (with different proteins or substrates) different mechan- the inhibited complex. This result suggests that the isms might be employed by the same . It is worth inhibitor complex represents a late intermediate of the noting that several members of the Gcn5/PCAF reaction just before peptide release. members contain a cysteine residue in the position Analysis of the tGcn5/inhibitor interface reveals that corresponding to C304 of Esa1, but these are not the pantetheine arm and pyrophosphate of CoA mediate mutationally sensitive and the cysteine is not strictly the most extensive protein contacts (Figure 2b), con- conserved within the Gcn5/PCAF family, as it is in the sistent with the CoA contacts in several other CoA- MYST family. Perhaps, the MYST proteins evolved to utilizing GNAT enzymes (Clements et al., 1999; retain this cysteine for specialized catalytic purposes. Clements and Marmorstein, 2003). These interactions Although not yet fully characterized, the p300/CBP likely provide a large degree of binding energy, but proteins appear to employ a catalytic mechanism that would be expected to exhibit a low degree of enzyme differs from the Gcn5/PCAF and MYST families of specificity. The lysine side chain and the isopropionyl HAT proteins. The observation that the bisubstrate linker of the inhibitor also make extensive van der inhibitor Lys-CoA, in which an acetyl bridge is instal- Waals interactions with the protein, but the peptide led between the substrate and CoA, is a potent region of the inhibitor only makes a handful of (IC50 ¼ 500 nM) p300 inhibitor suggested that the interactions with the protein. Together, the structure enzyme uses a ternary complex mechanism (Sagar of the tGCN5/H3-(Me)CoA-20 complex reveals that et al., 2004). However, neither the H3-CoA-20 nor small molecule compounds that optimize the interac- H4-CoA-20 peptide-CoA conjugates, where CoA is tions in the CoA pantetheine arm and pyrophosphate, linked analogously to 14 and 8 of the respective and the lysine and acetyl region, combined with histone peptides, are good p300 inhibitors (with IC50 enhanced interactions at the peptide–protein surface, values above 10 mM), while the H3-CoA-20 is a potent would provide a reasonable starting point for elaborat- (IC50 ¼ 360 nM) PCAF inhibitor (Poux et al., 2002). ing analogs of H3-(Me)CoA-20 with enhanced Gcn5/ Reciprocally, Lys-CoA is not a good PCAF inhibitor PCAF HAT specificity and potency. (with an IC50 of 200 mM). These results suggested that Although no other structures of HAT/inhibitor p300 might use a ternary complex mechanism that complexes have been reported, there have been reports differs from that of the Gcn5/PCAF family of HAT of the identification of other less potent HAT inhibitors. proteins. Interestingly, bisubstrate kinetic analysis of Several natural products have been reported including p300 is consistent with a ping-pong catalytic mechanism anacardic acid from cashewnut shell liquid (Varier et al., (Lau et al., 2000a). More work clearly needs to be 2004), and a polyisoprenylated benzophenone derivative performed on characterizing the catalytic mechanism of from Garcinia indica fruit rind called Garcinol that p300 and a structure of the catalytic domain of p300 or inhibits both the HAT activity of p300 and PCAF CBP would certainly be of significant use in this regard. (Balasubramanyam et al., 2004a) with a mid to low Nonetheless, it does appear that the catalytic mechanism micromolar IC50 value. In addition, a polyphenolic of the p300/CBP family differs from that of the Gcn5/ compound from Curcuma longa rhizome was shown to PCAF and MYST families of HAT proteins. Indeed, it be a specific inhibitor of p300/CBP HAT activity is quite surprising that these diverse HAT families may with an IC50 B25 mM. Interestingly, this inhibitor was employ different catalytic mechanisms that might reflect shown to be non-competitive with histone or Ac-CoA the distinct biology that these HAT families are (Balasubramanyam et al., 2004b), indicating that it associated with. As described above, it has already been binds to another site on p300/CBP. Structural studies demonstrated that the different catalytic mechanisms with these other HAT inhibitors may provide new between p300 and PCAF can be exploited to develop insights into the design of HAT-specific inhibitors. The HAT-specific inhibitors (Zheng et al., 2004). fact that the p300/CBP HAT proteins are translocated

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5532

Figure 2 Structure of an HAT/inhibitor complex. (a) Structure of tGcn5 bound to the H3-CoA-20 bisubstrate inhibitor. tGcn5 (PDB ID: 1M1D) is color-coded as described in Figure 1a and the bisubstrate inhibitor is shown in stick representation. (b) Interactions of H3-CoA-20 with tGcn5. The bisubstrate inhibitor is shown with the degree of protein interaction color-coded in gradation from extensive interaction (red) to minimal interaction (blue). HAT, histone acetyltransferase; tGcn5, Tetrahymena Gcn5.

in a subset of leukemias and act downstream of several structural homology with the other HDAC families and signal-transduction pathways suggests that p300/HAT use a distinct catalytic mechanism that is dependent on inhibitors may have therapeutic value. the oxidized form of adenine dinucleotide (NAD þ ) as a cofactor (Frye, 2000; Imai et al., 2000). HDAC activity is generally correlated with transcrip- tional repression and/or gene silencing (de Ruijter et al., Overview of HDACs 2003), and the importance of the class I and II enzymes as drug targets is illustrated by the fact that the aberrant Phylogenic analyses have subdivided HDACs into four recruitment of HDACs has been mechanistically linked families. Class I HDACs are homologous to yeast Rpd3 to malignancies in leukemias and lymphomas. Various and include human HDAC1–3 and -8; class II are small-molecule HDAC inhibitors that have shown homologous to yeast Hda1 and are further subdivided antitumor activity in model systems have advanced into into classes IIa (including human HDAC4, -5, -7 and -9) clinical trials (Lin et al., 2006; Gallinari et al., 2007), and and IIb (HDAC6 and -10) based on domain organiza- SAHA (suberoylanilide hydroxamic acid, ) tion and sequence homology. Class III HDACs are was recently shown to inhibit the growth of pancreatic named after yeast Sir2 and are also called sirtuins and cancer cells (Kumagai et al., 2007) and was recently include yeast Hst proteins 1–4 and human Sirtuins 1–7, approved by the Food and Drug Administration (FDA) and class IV are related to human HDAC11 (Gregoretti for the treatment of cutaneous T-cell lymphoma. There et al., 2004). Class I, II and IV HDACs share sequence is also a therapeutic interest in the sirtuins as they have and structural homology within their catalytic domains been linked to the correlation between caloric restriction and they share a related catalytic mechanism that does and life span extension in model organisms such as not require a cofactor but does require a zinc (Zn) metal yeast, worms, flies, fish and rodents; in mammals, sirtuin ion. In contrast, the sirtuins do not share sequence or activity has been linked to counteracting age-associated

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5533 diseases such as type II diabetes, obesity and neurode- 2007). Taken together, it is clear that additional studies generative disorders (Wood et al., 2004a, b; Kobayashi addressing the catalytic mechanism of the class I/II et al., 2005; Parker et al., 2005; Anekonda and Reddy, HDACs are still necessary, as these studies are likely to 2006; Baur et al., 2006). The association of sirtuins with influence our understanding of class I/II HDAC aging and diseases is discussed in greater detail in substrates and the development of improved class I/II this issue of Oncogene (Saunders and Verdin, 2007). HDAC inhibitors. Interestingly, several HDACs have been shown to Following the report of HDLP, two different groups target non-histone proteins for deacetylation as well. reported on the structure of the class I HDAC, HDAC8, For example, HDAC6 has been shown to deacetylate bound to several different inhibitors (Somoza et al., a- (Serrador et al., 2004), and SIRT1 has been 2004; Vannini et al., 2004). Not surprisingly, HDAC8 shown to deacetylate the p53 tumor suppressor protein shows a high degree of structural homology with (Luo et al., 2001; Cheng et al., 2003) and the forkhead HDLP. However, structural differences in the loops (FOXO) (Brunet et al., 2004) and surrounding the active site reveal a more accessible PPARg (peroxisome proliferator-activated receptor-g) active site for HDAC8; leading to the proposal that (Picard et al., 2004) transcription factors. Together, the HDAC8 might accommodate a variety of different different HDACs appear to have distinct substrate substrates. Another significant difference between the specificities leading to the regulation of different HDLP and HDAC8 structures is the identification of a biological activities. second monovalent metal (K þ or Na þ ) located near the active site in close proximity (7.0 A˚ )to the bound Zn ion. In addition, one of the reported HDAC8 structures shows a third monovalent metal Structural insights into catalysis and inhibitor binding by binding site away from the active site. Circular dichro- class I and II HDACs ism spectroscopy data suggest that K þ ions are required for the structural stability of HDAC8, and the structure Several structures have been reported for the class I and suggests that the K þ ion proximal to the Zn ion also II HDACs and they provide important insights into the plays a role in catalysis. The high degree of conservation mechanism of action of these enzymes as well as insights of the monovalent ligands among the HDAC proteins into inhibitor design. The first structure that was suggest that, in addition to Zn association, association reported was for a homologue of the class I HDACs with monovalent ions may be a conserved feature of from the hyperthermophilic bacterium Aquifex aeolicus class I HDAC proteins. called HDLP (histone deacetylase-like protein), which More recently, there was a report of a bacterial class shares 35.2% identity with human HDAC1 (Finnin II HDAC homologue FB188 HDAH (histone deacety- et al., 1999). This structure was determined both alone lase-like from Bordetella/Alcaligenes and bound to two hydroxamic acid inhibitors, TSA strain FB188) bound to the reaction product (tricostatin A) or SAHA. These structures reveal a and hydroxamic acid inhibitors (Nielsen et al., 2005). conserved deacetylase core domain consisting of an Sequence alignment of FB188 HDAH with other class I open a/b class of folds and a tubular active site pocket and II HDACs shows 30–35% sequence identity with containing the inhibitor and a bound Zn ion at the base the two HDAC domains of the class II human HDAC, (Figure 3a). The structural features present in the active HDAC6, and only about 20% identity with the class I site of HDLP suggest a mode of catalysis that has HDACs, HDLP and HDAC8. Nonetheless, a structural features of both metallo- and serine proteases in which superposition of FB188 HDAH with HDLP and the bound Zn atom mediates the nucleophilic attack HDAC8 shows that FB188 HDAH is as similar to the of a water molecule on the acetylated lysine sub- HDLP and HDAC8 as these proteins are to each other, strate, resulting in a tetrahedral oxyanion intermediate highlighting the high degree of structural homology (Figure 3b). The carbon–nitrogen bond of the inter- between the class I and II HDACs (Figure 3c). In mediate is then broken, with the nitrogen of the scissile particular, the active site Zn ion and both potassium bond primed to accept a proton from an Asp-His charge ions in FB188 HDAH were found at the equivalent relay, resulting in the formation of the acetate and lysine positions of the metal ions in the HDAC8 structure, and products. active site residues are essentially superimposable. Interestingly, a theoretical study suggests modifica- The most significant difference between the three class tions to the mechanism proposed by Finnin et al. (1999) I and II HDACs maps to the surface potential of the (Figure 3b) that differs from the previous proposal in proteins. FB188 HDAH is more acidic than the other the native protonation states of the His-Asp charge relay two HDACs. HDAC8 shows predominantly basic systems and the proposal that the deprotonation of patches, while HDLP shows an equal distribution of water is a distinct step in the mechanism (Vanomme- acidic and basic residues on the surface. Interestingly, all slaeghe et al., 2005). In addition, a recent report reveals the proteins appear to be acidic in the area around the that several HDACs also harbor esterase activity in entrance to the active site, although the degree of acidity addition to activity, and that the appears to vary (Figure 3d). In addition, the overall loop esterase activity involves the same active site but does structure of the different HDACs, including the loop not require an active site tyrosine (Figure 3b) that is structure around the active site, appears to differ as well, essential for aminohydrolase activity (Moreth et al., although it is not clear to what extent these different

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5534

Figure 3 Structure of class I/II HDACs. (a) Structure of HDLP bound to the SAHA inhibitor. Aquifex aeolicus HDLP (PDB ID: 1C3S) (blue) is shown bound to the SAHA inhibitor in stick representation (yellow) and a catalytic Zn þ 2 ion (green ball). (b) Catalytic mechanism of class I/II HDACs. The proposed catalytic mechanism for class I/II HDACs, as proposed based on the HDLP structure, is shown. (c) Superposition of class I and II HDACs. HDLP (PDB ID: 1C3S) (class I, slate-blue) is superimposed with HDAC8 (PDB ID: 1T64) (class I, purple) and FB188 HDAH (PDB ID: 1ZZ1) (class II, gray). The catalytic Zn þ 2 ion (yellow) and Na þ /K þ ions (orange) in HDAC8 and FB188 HDAH are also shown. (d) The active site entrance tunnel of class I/II HDACs. HDLP, HDAC8 and FB188 are shown in electrostatic surface representation. Blue, red and white are positive, negative and neutral electrostatic potential, respectively. The box highlights the acetyl-lysine substrate entrance tunnel. HDAC, histone deacetylase; HDAH, histone deacetylase- like amidohydrolase from Bordetella/Alcaligenes strain FB188; HDLP, ; SAHA, suberoylanilide hydroxamic acid.

loop structures reflect different crystal packing environ- Overall structure of sirtuins ments. Taken together, although it may be possible to exploit the slight differences in the electrostatic proper- The class III HDACs, or sirtuins, named after their ties and loop structures of the active site entrance of homology to the yeast Sir2 protein, do not have the different HDACs, it would appear that designing sequence or structural homology to the class I, II or HDAC-specific inhibitors for the class I and II HDAC IV HDACs and employ a different catalytic mechanism members might be particularly challenging. Although (Sauve et al., 2006). In addition to the acetyl-lysine some HDAC-specific inhibitors have been identified, substrate, sirtuins require the oxidized form of the such as tubacin, the mode of HDAC specificity cofactor NAD þ , for deacetylase activity (Sauve et al., exploited by this inhibitor is unknown (Hideshima 2001a; Jackson and Denu, 2002). These proteins convert et al., 2005). NAD þ and acetyl-lysine bearing histone substrates to

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5535 nicotinamide, deacetylated histone lysine and a novel intermediate, involving attack of the acetyl-lysine acetyl metabolite, 20-O-acetyl-adenosine diphosphate ribose group on the 10 carbon of the ribose ring (Sauve et al., (OAADPR) (Sauve et al., 2001a; Jackson and Denu, 2001b; Jackson and Denu, 2002) (Figure 4b). It is also 2002). The catalytic core domain of sirtuin proteins is well accepted that the nicotinamide inhibition reaction conserved from bacteria to human, although unlike occurs by base exchange of nicotinamide with an inter- bacteria and that typically have one or two mediate of the deacetylation reaction to reform b-NAD þ sirtuin proteins, multicellular organisms contain multi- through a transglycosidation reaction (Jackson et al., ple sirtuin proteins with variable N- and C-terminal 2003; Sauve and Schramm, 2003; Figure 4b). domains flanking the catalytic core. For example, There are two major issues of debate. This first issue contain five sirtuin proteins has to do with the mode of nicotinamide hydrolysis. One (Sir2 and Hst1–4) while there are seven human homo- model argues for an SN2-like reaction where nicotina- logues (SIRT1–7) (Frye, 2000). Interestingly, SIRT4 and mide hydrolysis from the b-face of the C10 position of 6 have been shown to catalyse ADP ribosylation instead the nicotinamide ribose ring is mechanistically linked to of deacetylation (Liszt et al., 2005; Haigis et al., 2006). nucleophilic attack on the a-face of the same carbon The molecular basis for why the SIRT4 and 6 members by the acetyl group of acetyl-lysine forming the of this family carry out related but different chemistry is O-alkylamidate intermediate (Hoff et al., 2006; Khan not known. and Lewis, 2006; Figure 4b). This model is supported by The structure of the catalytic core domain from a crystal structure of an unreacted ternary complex several sirtuin proteins from bacteria, archaea, yeast and between a bacterial sirtuin, acetyl-lysine and NAD þ , human have been determined using X-ray crystal- which shows the acetyl group of acetyl-lysine in position lography and reveals an elongated conserved catalytic to attack the a-face of the 10 carbon of the ribose ring core containing a structurally conserved (Hoff et al., 2006). However, the fact that the reaction domain characteristic of NAD þ /NADH-binding pro- has not turned over in the crystals argues that this teins and a smaller, more structurally variable domain ternary complex might not represent the catalytically containing a structural Zn ion (Marmorstein, 2004). A competent substrate configuration. A second model cleft between these two domains forms the binding sites argues for an SN1-like reaction where nicotinamide for the NAD þ and acetyl-lysine substrates (Figure 4a). hydrolysis occurs first followed by a rotation of the There is one reported structure of a full-length sirtuin nicotinamide ribose ring to permit acetyl-lysine attack protein, yeast Hst2, and this structure shows a homo- on the a-face of the 10 carbon of the nicotinamide trimeric structure that appears to be in an autoinhibited ribose ring. This mechanism necessitates formation of form. In particular, this structure shows that residues an oxocarbenium ion intermediate after hydrolysis of N-terminal to the catalytic domain sit within the acetyl- the nicotinamide and before nucleophilic attack of the lysine binding pocket of another protein subunit of the acetyl group of acetyl-lysine (Figure 4b). Consistent trimer and that a C-terminal helix sits within the NAD þ with this mechanism is a crystal structure of a ternary binding site of the same subunit (Zhao et al., 2004). complex of yHst2 with acetyl-lysine and a non-hydro- To what extent this feature is conserved within other lysable analogue of NAD þ , carba-NAD þ , in which the sirtuins is unknown; however, it appears unlikely since in-ring oxygen of the nicotinamide ribose is replaced regions N- and C- terminal to the conserved catalytic with a carbon atom (Zhao et al., 2004). This structure core show no sequence conservation among the sirtuin shows that the acetyl group of acetyl-lysine is hydrogen proteins. bonded to the 20- and 30-OH groups of the nicotinamide ribose and is not in position to carry out nucleophilic attack at the 10 carbon of the nicotinamide ribose ring. Instead, the carbonyl of a conserved asparagine residue, Catalytic mechanism of the sirtuins shown to be essential for nicotinamide hydrolysis, is in position to stabilize a positively charged oxocarbenium Although the reactants b-NAD þ and acetyl-lysine, and ion. Also consistent with this mechanism is a structure products: OAADPR, deacetylated lysine histones and of a ternary complex with yHst2, acetyl-lysine and the nicotinamide of the sirtuin deacetylation reaction are intermediate analogue, ADP-ribose that reveals that the well established, the detailed chemical mechanism has nicotinamide ribose ring rotates about 901 relative to its been a matter of debate in the literature. Nicotinamide orientation in carba-NAD þ and now places carbonyl has also been shown to be a non-competitive inhibitor of oxygen of acetyl lysine in position to carry out the sirtuin reaction, and the molecular basis for how this nucleophilic attach on the 10 carbon on the a-face of occurs has also been a matter of debate. The underlying the nicotinamide ribose ring (Zhao et al., 2004). These chemistry of the sirtuin deacetylation reaction as well as differing models for the mode of nicotinamide hydro- the nicotinamide inhibitory reaction has remained an lysis also have implications for the mode of nicotina- important issue, as it would influence the rational design mide inhibition. of small molecule sirtuin effectors. The following aspects The mode of nicotinamide inhibition of Sir2 proteins of the sirtuin reaction are generally accepted. First, for has been a second issue of debate in the literature. One the deacetylation reaction, the binding of acetyl-lysine is model argues that nicotinamide binds to the same required for nicotinamide hydrolysis and the reaction region, or so called ‘C-pocket’ that binds the nicotina- proceeds through formation of an O-alkylamidate mide group of NAD þ (Avalos et al., 2004) (Figure 4c).

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5536

Figure 4 Overall structure of class III (sirtuin) HDACs. (a) Structure of the sirtuin catalytic domain. The catalytic domain of yeast Hst2 (PDB ID: 2OD2) (green) bound to an acetyl-lysine 16 histone H4 peptide (blue, stick representation) and a non-hydrolysable NAD þ analogue (carba-NAD þ , orange) is shown. A structural Zn þ 2 ion is shown as a blue ball. (b) Catalytic mechanism of class III (sirtuin) HDACs. The proposed SN1- and SN2-like catalytic mechanisms for the sirtuin deacetylation reaction are shown with the acetyl-lysine (green), NAD þ (red) and protein residues in yeast Hst2 (blue) color-coded. (c) The nicotinamide inhibitory site on sirtuins. A surface representation of the yeast Hst2 active site is shown with acetyl-lysine (blue), the non-hydrolysable NAD þ analogue, carba-NAD þ (purple), an analogue of the transition state, ADP-HPD (green) and nicotinamide (orange). The C and D nicotinamide binding sites of NAD þ and free nicotinamide for inhibition/base exchange are indicated, respectively. HDAC, histone deacetylase; NAD þ , oxidized form of nicotinamide adenine dinucleotide.

Consistent with this hypothesis is the general micro- to NAD þ or ADP-ribose and a Termatoga maritime reversibility of chemical reactions and structures of sirtuin protein (Sir TM) bound to acetyl-lysine in the Archaeoglobus fulgidus sirtuin protein (Sir2-Af2) bound presence of high concentrations of nicotinamide that

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5537 shows nicotinamide bound in the C-pocket (Avalos including its use as a chemotherapeutic agent (Manson et al., 2005). However, neither Sir2-Af2/NAD þ , Sir2- et al., 2005). Interestingly, although cell-based studies Af2/ADP-ribose nor Sir TM/acetyl-lysine is the enzy- have clearly shown a SIRT1-dependant activation in matic complex to which an inhibitory nicotinamide response to resveratrol (Howitz et al., 2003; Wood et al., molecule is expected to bind. A second model argues 2004a; Baur et al., 2006), the biochemical activation of that the inhibitory nicotinamide molecule binds to a sirtuin proteins has been a matter of debate and will nearby ‘D-pocket’ that is distinct from the C-pocket require further study (Howitz et al., 2003; Borra et al., (Figure 4c). This model was initially based on a 2005; Kaeberlein et al., 2005). The structural basis for structure of yHst2 in complex with lysine-16 histone how resveratrol or the other identified effectors of the H4 and a reaction intermediate analogue, ADP-ribose, sirtuin enzymes work is still unclear and will require that modeled an incoming nicotinamide molecule within structural characterization of relevant sirtuin/effector the D-pocket (Zhao et al., 2004). More recently, a complexes. crystal structure of nicotinamide within the D-pocket was obtained by socking nicotinamide into crystals containing yHst2, lysine-16 histone H4 and reaction Perspectives intermediate analogue designed to mimic the oxocarbe- nium ion, ADP-HPD (Sanders et al., 2007). In addition, This review illustrates the complexity, structural diversity biochemical studies showed that mutagenesis of the and mechanistic ingenuity of the enzymes that catalyse residues in the D-pocket does not significantly affect acetyl-transfer on histones. We now have structures and NAD þ binding while having more significant effects on the enzymatic characterization of several of the protein the Ki for nicotinamide inhibition. This same study subfamilies that mediate this acetyl transfer. Together, revealed that a mutation in the C-pocket of an this has provided a wealth of information on the asparagine residue that contacts nicotinamide in NAD þ chemistry of these enzymes that can now be exploited þ decreases NAD binding but does not alter the Ki for to design small molecule compounds that might effect the nicotinamide inhibition. Based on these results, it activity of these enzymes in ways that may be useful for appears that the D-pocket plays a more significant role the treatment of human disease and cancer in particular. for nicotinamide inhibition and base exchange by the Despite these efforts, however, there are still unresolved sirtuin proteins, although additional experiments will issues and more recent accumulating data that suggest likely have to be carried out to resolve this discrepancy. that there is still much work ahead. With regard to the HAT enzymes, do the different subfamilies use a conserved ternary complex catalytic Sirtuin effectors mechanism or do they use different catalytic mechan- isms that are perhaps modulated in a substrate specific The liganded sirtuin structures described above point to way? How different is the structure of the p300/CBP the targeting of either the acetyl-lysine or NAD þ HAT from the Gcn5/PCAF and MYST HATs and binding site for protein inhibition. However, since many what is the relationship of their catalytic mechanisms? other NAD þ utilizing enzymes use a structurally Resolving issues such as these may be at the heart of conserved Rossmann fold to bind NAD þ , targeting designing HAT-specific inhibitors that may be used for the NAD þ site might not be sufficiently specific for the targeting, for example, chromosomal translocations in sirtuin proteins. The acetyl-lysine target site is, however, leukemia that involve the p300/CBP and MYST family a viable option for the design of sirtuin inhibitors, of HAT proteins. particularly since the acetyl-lysine binding site shows With regard to the class I and II HDACs, although significant differences with the acetyl-lysine binding site vorinostat has been approved by FDA for treating T-cell- of the class I/II HDACs. Consistent with targeting the lymphoma treatment, it will be interesting to follow the acetyl-lysine site of the sirtuin proteins for inhibition, a development of other class I/II HDAC inhibitors in recent report describes the identification of the sirtuin current clinical trials to determine if these general inhibitor cambinol that shows competitive binding with inhibitors will be useful or if more specific class I/II acetyl-lysine (Heltweg et al., 2006). The targeting of the HDAC inhibitors need to be developed. If the latter is the nicotinamide inhibitory, D-pocket, may also represent a case, it will be helpful to obtain more structural fruitful avenue of investigation. Since nicotinamide is a information of other class I/II HDACs, where HDAC1 known physiological regulator of sirtuin enzymes, relief and -2 appear to be at the top of the wish list. Additional of nicotinamide inhibition would be expected to result in enzymatic studies will also be required to resolve some of the net activation of sirtuin proteins. Molecules that the disparities underlying the catalytic mechanism and block the D-pocket and the nearby C-pocket may also substrates of the class I/II HDACs, as resolving such serve as potent and specific Sir2 inhibitors. issues are likely to contribute to the further development To date, several sirtuin effectors have been described of class I/II HDAC inhibitors. There is a strong case to including the inhibitors, sertinol (Kiviranta et al., 2006), be made for the development of sirtuin effectors for the splitomicin (Posakony et al., 2004) and a group of indole treatment of age-associated diseases such as type II analogues and the activator resveratrol (Biel et al., diabetes, obesity, neurodegenerative disorders and can- 2005). The resveratrol activator has received much cer. Structural information of sirtuin proteins bound to attention because of its many reported health benefits effectors may be useful for structure-based design efforts

Oncogene Structure and chemistry of HATs and HDACs SC Hodawadekar and R Marmorstein 5538 and it would appear that targeting of the nicotinamide Notes added in proof inhibitory C- or D-pockets for the development of both sirtuin activators and inhibitors might be particularly During the revision of this review three relevant fruitful for the development of broad-based sirtuin manuscripts were published: (1) Nielsen et al. (2007) effectors. It is unclear at this point if sirtuin-specific reported on the structure of a bacterial class II HDAC effectors will be desirable, but if this is the case, bound to a trifluromethylketone inhibitor revealing the additional structures of the various homologues may be first structure of an HDAC bound to a non-hydro- useful. Although there are still a lot of questions that xamate HDAC inhibitor; (2) Schuetz et al. (2007) remain underlying the chemistry and small molecule reported on the structure of SIRT5 bound to the regulation of these fascinating enzymes that mediate inhibitor suramin revealing the first structure of a acetyl transfer, one thing remains clear – the acetylation sirtuin/inhibitor complex; and (3) Smith and Denu status of histones represents an important signal-trans- (2007) reported on a study demonstrating nucleophilic duction pathway that is critical for normal cell processes participation of acetyl-lysine in NAD þ cleavage, sup- and will remain an important target for the treatment of porting an SN2-like catalytic mechanism for Sir2 human diseases such as cancer. proteins.

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