Structure, Vol. 12, 1325–1334, July, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.04.012 Structural Snapshots of Human HDAC8 Provide Insights into the Class I Deacetylases

John R. Somoza,1,3,* Robert J. Skene,2 types with HDAC inhibitors (HDIs) does not cause a Bradley A. Katz,1 Clifford Mol,2 Joseph D. Ho,1 global increase in transcription. Instead, only a small Andy J. Jennings,2 Christine Luong,1 percentage of the genome is affected (Glaser et al., Andrew Arvai,2 Joseph J. Buggy,1 Ellen Chi,2 2003; Van Lint et al., 1996). Treatment with HDIs causes Jie Tang,1 Bi-Ching Sang,2 Erik Verner,1 tumor cells to cease growth and to either differentiate Robert Wynands,2 Ellen M. Leahy,1 or become apoptotic. These findings have led to interest Douglas R. Dougan,2 Gyorgy Snell,2 Marc Navre,2 in the use of HDIs as antitumor agents, resulting in sev- Mark W. Knuth,2,4 Ronald V. Swanson,2,4 eral small molecules that are currently in clinical trials Duncan E. McRee,2,4 and Leslie W. Tari2,3,* for oncology indications (Johnstone, 2002). Moreover, 1Celera a number of studies have shown potential application 180 Kimball Way of HDIs in the treatment of neurodegenerative disorders South San Francisco, California 94080 (Hockly et al., 2003) and in the treatment of genetic 2 Syrrx, Inc. disorders such as ␤-thalassemia (Witt et al., 2003). 10410 Science Center Drive Two related classes of eukaryotic zinc-containing San Diego, California 92121 HDACs have been recognized based upon sequence similarity: class I (HDAC1, 2, 3, and 8) and class II (HDAC4, 5, 6, 7, 9, and 10) (Johnstone, 2002; Gao et al., Summary 2002). Another HDAC, HDAC11, lies at the boundary between the two classes. Both class I and II HDACs Modulation of the state of plays possess well-conserved deacetylase core domains of a pivotal role in the regulation of expression. approximately 400 amino acids and apparently identical Histone deacetylases (HDACs) catalyze the removal Zn2ϩ-dependent catalytic machinery (Figure 1). Class I of acetyl groups from lysines near the N termini of HDACs are ubiquitously expressed and are primarily histones. This reaction promotes the condensation of located in the nucleus, while the class II can chromatin, leading to repression of transcription. shuttle between the nucleus and cytoplasm and demon- HDAC deregulation has been linked to several types strate tissue-specific expression (Johnstone, 2002; of cancer, suggesting a potential use for HDAC inhibi- Marks et al., 2001; Vigushin and Coombes, 2002). tors in oncology. Here we describe the first crystal HDAC8 is a 377 residue class I that lies close structures of a human HDAC: the structures of human to the phylogenetic boundary between the class I and HDAC8 complexed with four structurally diverse hy- class II HDACs, and maps to the X (Hu et droxamate inhibitors. This work sheds light on the al., 2000; Buggy et al., 2000; Van den Wyngaert et al., catalytic mechanism of the HDACs, and on differences 2000). The physiological role played by HDAC8 is gradu- in substrate specificity across the HDAC family. The ally being elucidated. Lee et al. (2004) have shown that structure also suggests how phosphorylation of Ser39 HDAC8 is phosphorylated in vivo by cyclic AMP-depen- affects HDAC8 activity. dent protein kinase A (PKA), and that phosphorylation decreases the activity of HDAC8 and leads to the Introduction hyperacetylation of histones H3 and H4. This work indi- cates that HDAC8, under the regulation of PKA, plays Acetylation of the core histones of nucleosomes strongly an important role in determining the acetylation state of influences gene transcription (Khochbin et al., 2001; histones. Also, recent evidence suggests that HDAC8 Johnstone, 2002). Two families of enzymes modulate may play a role in one of the most frequent types of acute histone acetylation: histone acetyl (HATs) myeloid leukemia (AML). Inversion(16) is a chromosomal and histone deacetylases (HDACs), which catalyze the translocation that frequently occurs in AML and results addition and removal, respectively, of acetyl moieties in a protein product associated with abnormal transcrip- from the ⑀-amino groups of lysines near the amino ter- tional repression. The inversion(16) protein product as- mini of histones. Histone deacetylation promotes the sociates with HDAC8, and the repression that is medi- condensation of chromatin which, in turn, promotes ated by this complex is sensitive to HDAC inhibitors transcriptional repression (Struhl, 1998). The HDACs, in (Durst et al., 2003). particular, the class I HDACs, play an important role in We present the first three-dimensional structures of gene silencing as they are recruited to key locations a mammalian HDAC, including crystal structures of in nucleosomes through their interactions with tran- HDAC8 complexed to trichostatin A (TSA), suberoylani- scription complexes such as Sin3 and NcoR (Ng and lide hydroxamic acid (SAHA), 4-dimethylamino-N-(6- Bird, 2000). Interestingly, treatment of a variety of cell hydroxycarbamoyethyl)benzamide-N-hydroxy-7- (4-dimethylaminobenzoyl)aminoheptanamide (MS-344), and 5-(4-methyl-benzoylamino)-biphenyl-3,4Ј-dicar- *Correspondence: [email protected] (J.R.S.), ltari@syrrx. boxylic acid 3-dimethylamide 4Ј-hydroxyamide (CRA-A) com (L.W.T.) 3 These authors contributed equally to this work. (Figure 2A; Table 1). These inhibitors are active against 4 Present address: ActiveSight, 4045 Sorrento Valley Boulevard, San class I and class II HDACs, and have the ability to induce Diego, California 92121. multiple effects within cells, including cell differentiation, Structure 1326

Figure 1. Sequence Alignment of the Class I HDACs and HDLP Residues that are identical across the enzymes are highlighted in blue, and the overall identities are 40%, 41%, 41%, and 31% between the sequence of HDAC8 and the sequences of HDAC1, HDAC2, HDAC3, and HDLP, respectively. The key catalytic residues in HDAC8 are also conserved in the class II HDACs. The sequences corresponding to the C-terminal domains of HDACs 1, 2, and 3 are not shown. induction of arrest, and suppression of tumor and encompass the residues that form the enzyme’s growth (Jung et al., 1999; Almenara et al., 2002; Richon and catalytic machinery. A DALI search (Holm et al., 1998; Yoshida et al., 1990a, 1990b). SAHA is cur- and Sander, 1994) of the Protein Data Bank revealed rently in phase II clinical trials for the treatment of solid that HDAC8 has a core architecture similar to that found tumors and hematologic malignancies. The HDAC8 in two enzymes: , which catalyzes the conver- structures shed light on the catalytic mechanism of the sion of arginine to ornithine (Kanyo et al., 1996), and an class I and class II HDACs and provide new information HDAC-like protein (HDLP) from the hyperthermophilic on substrate binding in the class I enzymes. These struc- bacterium Aquifex aeolicus (Finnin et al., 1999), an tures also provide a framework for the identification of enzyme with no known function but which shares ap- novel HDAC inhibitors. proximately 31% sequence identity with HDAC8. The ␣-carbons of the HDAC8 and arginase structures super- Results and Discussion impose with a root-mean-square deviation of 3.5 A˚ over two-thirds of the residues, primarily in the vicinity of the Overall Structure of HDAC8 central ␤ sheet. The ␣-carbons of the HDAC8 and HDLP HDAC8 comprises a single ␣/␤ domain that includes an structures can be overlaid with root-mean-square devia- eight-stranded parallel ␤ sheet sandwiched between 13 tion of 2.2 A˚ over 90% of the residues. While the folds of ␣ helices (Figure 2B). While approximately half of the HDAC8 and HDLP are similar, the two protein structures amino acids are contained in canonical secondary struc- differ dramatically in the outer portions of their active ture elements, the other half of the residues in HDAC8 sites, and in the conformations and topology of their form loops that link the various elements of regular sec- last 50 amino acid residues (Figure 2C). ondary structure. The long loops emanating from the Comparisons of the structures of HDAC8 and HDLP, C-terminal ends of the strands of the core ␤ sheet form coupled with analysis of the sequence alignments of the a substantial portion of the volume of the entire enzyme class I HDACs and HDLP (Figure 1), reveal that HDAC8 Structures of Human HDAC8 1327

Figure 2. Overall Structure of HDAC8 and the Structures of the Four Hydroxamate Inhibitors (A) Chemical structures of the four inhibitors used in this study. From left to right the inhibitors are TSA, SAHA, CRA-A, and MS-344. (B) Ribbon diagram of the HDAC8:TSA complex showing the HDAC8 fold, as well as the inhibitors and the zinc ion. The TSA molecules are shown in blue and pink, and the zinc ion is shown in orange. (C) Stereo diagram showing superposition of the HDAC8:TSA structure (cyan) and the structure of apo HDLP (red) (Finnin et al., 1999). The L1 loops of HDAC8 and HDLP are shown in green and yellow, respectively. Structure 1328

Table 1. Data and Refinement Statistics HDAC8:TSA HDAC8:MS-344 HDAC8:SAHA HDAC8:CRA-A Data

Space group P43 P3121 P3121 P212121 Cell parameters (A˚ ) 81.0, 81,0, 114.2 80.6, 80.6, 105.5 80.8, 80.8, 105.6 82.6, 92.7, 98.8 Resolution range (A˚ ) 59.0–1.9 69.0–2.3 70.0–2.9 27.88–2.20 Total reflections 241,421 92,000 46,761 148,248 Unique reflections 54,547 16,130 8164 37,437 Completeness (last shell) (%) 99 (99) 94 (93) 95 (95) 91 (62)

Rsym(I) (last shell) (%) 5.8 (54.4) 5.1 (54.6) 7.6 (54.5) 9.2 (34.6) ϽIϾ/Ͻ␴(I)Ͼ (last shell) 23.8 (2.5) 30.0 (2.2) 16.8 (4.0) 6.4 (1.6)

Model Molecules in asymmetric unit 2 1 1 2 Number of water molecules 681 137 0 545 Other ligands 2 Zn2ϩ,3Ca2ϩ,4Naϩ 1Zn2ϩ,2Naϩ 1Zn2ϩ 2Znϩ2,2Naϩ Resolution used for refinement (A˚ ) 59.0–1.9 69.0–2.3 70.0–2.9 7.0–2.2 Sigma cutoff (F/␴(F)) 0.0 0.0 0.0 1.1

R factor (Rfree, 5% of data) 17.2% (22.1%) 21.2% (27.4%) 24.9% (31.0%) 23.2% (30.6%) Rms deviations from ideal geometry Bonds (A˚ ) 0.010 0.009 0.010 0.018 Angles (Њ) 1.27 1.14 1.28 3.79 is structurally and, by implication, functionally unique in In each of the structures presented here, the tunnel is several key aspects. The main feature distinguishing filled by the aliphatic chain of the inhibitor. During the HDAC8 from the other class I enzymes is the absence deacetylation reaction, the tunnel is presumably occu- of a 50–111 amino acid C-terminal domain that extends pied by the four methylene groups of the acetylated from the catalytic domain (which ends at residue 377 in lysine. The walls of the tunnel, formed by F152, F208, HDAC8). In HDACs 1–3, the C-terminal domains are used H180, G151, M274, and Y306, are primarily hydrophobic to recruit the enzymes to protein complexes that modu- (Figures 3A and 3B). These residues are conserved late their enzymatic activities and localization (Ayer, across the class I HDACs, with the exception of M274, 1999). Additionally, the activities of class I HDACs are which is a leucine in the other family members (Figure 1). regulated by posttranslational modifications to the The catalytic machinery lies at the end of the hy- C-terminal extension, such as sumoylation (David et al., drophobic tunnel. The centerpiece of this region is a 2002) and phosphorylation (Tsai and Seto, 2002), which zinc ion that is bound to carboxylate oxygens of D178 may affect their catalytic activities via an allosteric and D267, and to the N␦1 atom of H180 (Figure 3A and mechanism and/or by altering their complex forming 3C). Two other coordination sites are occupied by the capabilities. Given this difference between HDAC8 and carbonyl and hydroxyl oxygens of the inhibitors’ hydrox- the other class I enzymes, it seems likely that HDAC8 amate. It seems likely that when the natural substrate either does not require recruitment to protein complexes binds, these coordination points would be occupied by to function or that its recruitment utilizes entirely differ- the carbonyl oxygen of the acetyl moiety and by a water ent regions of the protein surface. A second feature molecule, respectively. The HDAC8 and HDLP struc- distinguishing HDAC8 from HDLP and all other identified tures, as well as mutagenesis (Hassig et al., 1998; Ka- class I HDACs is the size and composition of the N-terminal dosh and Struhl, 1998) and sequence conservation data L1 loop (residues 30–36, Figures 1 and 2C). In both (Figure 1), suggest important structural/catalytic roles HDAC8 and HDLP, the L1 loop lines a large portion of for the bound zinc ion, for histidines 142 and 143, and one face of the active site pocket and extends to the for Y306. D176 and D183 are also likely to be important protein surface (Figure 2C). However, the conformations for catalysis. The carboxylate moieties of these aspar- of the L1 loops in HDAC8 and HDLP are dramatically tates form hydrogen bonds with the N␦1 atom of each different, and the HDAC8 loop is two residues shorter histidine, similar to the Asp-His charge relay system than its counterpart in HDLP (Figures 1 and 2C). The seen in serine proteases that is known to increase the result is that HDAC8 has a wider active site pocket with basicity of the imidazole N⑀2 atom. a larger surface opening. Interestingly, the sequence There is a second metal buried in the alignments shown in Figure 1 suggest that HDLP and interior of the protein in the vicinity of the active site, HDACs 1–3 possess structurally homologous L1 loops, approximately 7 A˚ from the zinc, which in the HDAC8 and that HDAC8 is the structural outlier in the vicinity structures is occupied by a sodium ion (Figure 3A). The of the L1 loop in the active site pocket. The functional metal is bound in an octahedral geometry by six oxygen implications of this observation are discussed in a later ligands in the protein: the carboxylate of D176, the side section. chain hydroxyl oxygen of S199, and the backbone car- bonyl oxygens of residues 176, 178, 180, and 200. The The Structure of Active Site and Substrate coordination distances between the sodium ion and co- Binding Tunnel ordinating groups varies from 2.5 to 3.0 A˚ . These dis- The HDAC8 active site consists of a long, narrow tunnel tances and the octahedral coordination geometry are leading to a cavity that contains the catalytic machinery. consistent with what is commonly observed for sodium Structures of Human HDAC8 1329

Figure 3. Interactions of HDAC8 with Inhibi- tors and Substrate (A) Stereo figure showing key residues in the HDAC8 active site, two TSA molecules (blue and pink), the zinc (orange), and the sodium (purple). Potential hydrogen bonds are de- picted as dashed lines.

(B) Fo Ϫ Fc simulated annealing omit maps showing the electron density for the two mol- ecules of TSA. The maps were contoured at 2.5␴ and are shown within 1.8 A˚ of the TSA molecules. (C) Schematic diagram showing the binding of the hydroxamate inhibitors to HDAC8. (D) Schematic diagram showing the pro- posed model of how the acetylated lysine would interact with the HDAC catalytic ma- chinery.

ions in proteins (Harding, 2002). The role of the bound The sodium may modulate the basicity of the H142 N⑀2 sodium atom is not clear, although a sequence compari- nitrogen through its interaction with D176. Alternatively, son of the class I HDACs suggests that the binding site this ion may play a structural role in stabilizing the geom- for this ion is conserved across this group of enzymes. etry of the active site, as it coordinates and stabilizes a Structure 1330

Figure 4. The Surface of HDAC8 Is Malleable and Changes to Accommodate Different In- hibitors

(A) and (B) C␣ traces show how the L1 loop moves between the HDAC8:MS-344 (A) and HDAC8:TSA (B) complexes. In (B), the L1 loop of both structures is shown to facilitate the comparison. In the HDAC8:TSA complex, the K33 side chain is disordered and is not shown. The ␣-carbon of K33 moves by 6 A˚ toward the active site pocket in the MS-344 and SAHA (data not shown) complexes, so that the methylene carbons of the K33 side chain interact with the catalytic pocket lining F152 residue, and the loop occludes the sec- ond pocket that is present in the TSA and CRA-A (data not shown) complexes. (C) Solvent accessible surfaces of the active site regions of the HDAC8:TSA, HDAC8:MS- 344, HDAC8:SAHA, and HDAC8:CRA-A com- plexes. The MS-344 and SAHA complexes only possess a catalytic pocket, while the TSA and CRA-A complexes each possess an adjacent binding pocket.

tight loop on the protein interior that presents key resi- binding both the nucleophile and the substrate, and (2) dues to the active site. by polarizing the carbonyl of the acetyl-lysine (in concert with the hydroxyl of Y306), increasing its electrophilicity. Implications of the Active Site Structure The charged tetrahedral intermediate resulting from the for the Catalytic Mechanism nucleophilic attack could be stabilized through interac- The HDAC8 and HDLP structures place constraints on tions with the zinc and interactions with the side chain possible catalytic mechanisms employed by the class hydroxyl of Y306. The collapse of the tetrahedral inter- I and II HDACs. A likely first step in the deacetylation mediate would yield the acetic acid and lysine products. reaction is a nucleophilic attack on the carbonyl carbon Finnin et al. (1999) have suggested that H143 protonates of the substrate. Given that none of the residues in the the lysine after the carbon-nitrogen scissile bond vicinity of the substrate are reasonable nucleophiles, it breaks. In the HDAC8 structures, the histidine is approxi- seems probable that this function is carried out by a mately 4 A˚ from where we would expect the amino group zinc-bound water molecule (Figure 3D). In the HDAC8 of the lysine to end up during catalysis, suggesting that complex structures presented here, the hydroxyl group the protonation of the histidine may be water mediated. of the hydroxamic acid is likely a structural mimetic of the catalytic water. The zinc would lower the pKa of a The Structures of the Four HDAC8:Inhibitor water proton, making the water more nucleophilic. The Complexes Show that the Surface of HDAC8 nucleophilicity of the water would probably also be in- in the Vicinity of the Active Site creased through hydrogen bonds with the N⑀2 of H142 Is Extremely Malleable and H143. In addition to affecting the pKa of the water, A comparison of the structures of the four HDAC8:inhibi- the zinc could facilitate the deacetylation reaction in two tor complexes presented here reveals considerable ways: (1) by reducing the entropy of the reaction by structural differences in the protein surface in the vicinity Structures of Human HDAC8 1331

of the opening to the active site (Figure 4). These differ- ences suggest that this region is highly malleable and is able to change to accommodate binding to a variety of different ligands. From a physiological point of view, this flexibility suggests that HDAC8 might be able to bind to acetylated lysines that are presented in a variety of structural contexts. The differences that are observed in the HDAC8 sur- face around the active site are primarily mediated by the L1 loop (S30-K36) (Figures 2C, 4A, and 4B). Among the four complexes, the position of the ␣-carbon of K33 in the L1 loop varies by as much as 6 A˚ with respect to the protein core. In the HDAC8:CRA-A complex, the L1 loop peels away from the protein, exposing a deep binding groove immediately adjacent to the acetyl-lysine binding site (Figure 4C). This groove measures approxi- mately 13 ϫ 5A˚ and reaches a depth of about 15 A˚ . Figure 5. Relative Positions of the HDAC8 Active Site and the Phos- F152 and Y306, which line the catalytic pocket, also line phorylation Site on Serine 39 the second pocket. In the HDAC8:TSA complex, the Solvent-accessible surface of the structure of HDAC8:TSA showing conformation of the protein changes in two ways with the TSA molecule binding in the active site (green), the TSA binding respect to the HDAC8:CRA-A complex: (1) several of in the second site (cyan), and serine 39 (red). the residues that line the groove collapse inwards, and (2) loop L2 (residues 97–103), which is poorly ordered in the HDAC8:CRA-A structure, becomes ordered due to about HDAC8 flexibility to the other class I HDACs. Se- a ␲-␲ stacking interaction between the dimethyl aniline quence alignments of HDAC8 with the remaining class moiety of the active site bound TSA and Y100. The effect I HDACs suggest that the other enzymes in the family of these changes is to close off a large part of the second possess longer L1 loops, structurally similar to the loop binding groove, leaving a narrower second pocket. Sur- observed in HDLP (Figure 1). Based on the apparent prisingly, this second cavity is filled by a second TSA rigidity of the HDLP active site described above, it is molecule (Figures 3A and 4C), which appears to have reasonable to assume that HDAC1, 2, and 3 also pos- induced the collapse of the pocket to optimize protein- sess more conformationally static active sites than ligand interactions. The second TSA molecule is rotated HDAC8. However, a definitive and thorough picture of by approximately 150Њ with respect to the TSA bound active site flexibility and function of the other HDACs in the catalytic pocket, and inserts its dimethyl aniline will require an understanding of how they each react to head group into the second pocket where it interacts accommodate different bound ligands, or ligands bound with several hydrophobic residues (Figure 3A). The hy- in different environments. The identification of potent drophobic linker of the second TSA molecule sticks out and selective HDAC inhibitors will require similar infor- of the second pocket and interacts with the ac- mation. Potent inhibitors can be identified by targeting tive site bound TSA (Figures 3A and 4C). In the the well-conserved catalytic machinery and binding HDAC8:MS344 and HDAC8:SAHA structures, the L1 pocket. However, the identification of compounds that loop moves toward the active site and positions K33 so specifically inhibit a single member of the HDAC family that the side chain methylene groups pack against the will be aided by a clear picture of the structure and F152 side chain, resulting in the occlusion of the second flexibility of the surface of each HDAC. pocket (Figure 4). The fact that HDAC8 is capable of exposing a large second cavity suggests that the en- The HDAC8 Structure Suggests How the Protein’s zyme could deacetylate lysine residues that are sur- Activity Is Regulated by Phosphorylation at Serine 39 rounded by bulky amino acids, or in environments where Lee et al. (2004) have shown that serine 39 of HDAC8 the lysine residues are partially buried. is phosphorylated in vivo and in vitro by PKA, and that None of the HDLP structures exhibits an exposed phosphoryation leads to a decrease in the enzyme’s second binding site, presumably because HDLP has a activity. S39 lies at the surface of HDAC8, roughly 20 A˚ longer L1 loop than HDAC8 (Figures 1 and 2C). Analysis from the opening to the HDAC8 active site and roughly of the HDLP structure indicates that it also possesses 13 A˚ from the opening to the second binding site (Figure a deep pocket adjacent to the active site, but that the 5). S39 resides in a surface pocket comprising several pocket is occluded by the longer loop. The conformation hydrophobic residues, including V25, A38, F336, and of this loop is virtually identical in the three HDLP struc- part of H42. In addition, the hydroxyl group of the serine tures (Finnin et al., 1999), suggesting that the surface is flanked on opposite sides of the pocket by two acidic of HDLP is less malleable than that of HDAC8 around residues: E335 and D29. The D29 side chain hydrogen the binding site pocket. The apparent reduced flexibility bonds with the serine hydroxyl group. It is also interest- in the HDLP surface may also explain why it is less ing to note that a lysine residue, K36 has its side chain efficient in assays (Finnin et al., positioned between S39 and the active site pocket, 5 A˚ 1999). from S39. This places K36 within range to directly inter- Using the structural comparisons between HDAC8 act with phosphorylated S39. It is clear from the HDAC8 and HDLP, we can extrapolate the information gained structure that the placement of a phosphate in this envi- Structure 1332

Figure 6. Schematic View of the Solid-Phase Synthesis of CRA-A

ronment would lead to a major structural disruption of BRL). Expression using this vector results in a fusion protein with this region of the surface. an N-terminal poly-histidine affinity tag. Recombinant baculovirus There are two plausible ways in which a disruption of incorporating the HDAC gene was generated by transposition with the Bac-to-Bac system (Invitrogen), and high-titer viral stocks were the structure in the vicinity of S39 might negatively affect generated by infection of Spodoptera frugiperda Sf9 cells. The ex- the protein’s activity. One possibility is that the structural pression of recombinant protein was carried out in either Tri- rearrangement provoked by the phosphorylation ex- choplusia ni Hi5 cells (Invitrogen) or in Sf9 cells. tends to the HDAC8 active site, thus disrupting activity. This possibility seems fairly likely given that S39 inter- Purification acts with structural elements that extend into the active The protein used for the TSA, SAHA, and MS344 complexes was site, including K36, which forms part of the conforma- isolated from cell extracts by passage over ProBond (Invitrogen) tionally mobile L1 active site loop. Given the flexibility resin and concentrated to 10–16 mg/ml. Protein samples were stored at 4ЊC in 25 mM Tris-HCl (pH 7.6), 250 mM NaCl, and 0.1 mM that has been observed for this region of the protein, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). S39 phosphorylation may induce a conformation of the To purify the protein used to crystallize the HDAC8:CRA-A com- L1 loop that precludes catalytically competent substrate plex, infected Sf9 cells were harvested 72 hr post infection and binding. A second possibility is that S39 forms part of stored at Ϫ80ЊC. Frozen cells were lysed by thawing at room temper- the surface that interacts with the target histone and ature in a buffer consisting of 50 mM Tris-HCl (pH 7.9), 200 mM that the disruption of the HDAC8-histone interaction in- NaCl, 1 mM TCEP, and a protease inhibitor mix (EDTA free) (Roche terferes with activity. Molecular Biochemicals). The lysate was cleared by centrifugation, and loaded onto Ni-NTA resin (Qiagen) which had been equilibrated with 20 mM Tris-HCl (pH 8.0), 30 mM imidazole, 1 M NaCl, and 0.25 Conclusions mM TCEP. Bound protein was eluted with 20 mM Tris-HCl (pH 7.0), Our results on human HDAC8 provide the first structure 500 mM NaCl, 250 mM imidazole, and 0.25 mM TCEP. The eluted of a member of the class I or class II histone deacety- protein solution was concentrated on YM10 membrane (Millipore) lases. The HDAC8 structure places strong constraints and further purified on a Sephacryl S 200 gel filtration column. The on how catalysis occurs in this family of enzymes, and final elution buffer was 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and leads to a proposed catalytic mechanism that is similar 0.25 mM TCEP. to the mechanism proposed for HDLP. The structures also suggest that malleability of the HDAC8 surface Synthesis of CRA-A The synthetic procedure used for the preparation of CRA-A is out- plays an important role in its activity, and provide insight lined in Figure 6. Solid-phase synthesis using commercially available on how the phosphorylation of serine 39 affects histone hydroxylamine Wang resin proved to be a straightforward method deacetylation. Finally, these HDAC8 structures provide for the parallel preparation of numerous analogs. A series of amide a picture of how inhibitors that are currently in clinical coupling reactions followed by the liberation of the hydroxamic acid trials bind to the enzyme, and provide a starting point from the resin afforded materials of the same or better quality than for the design of novel HDAC inhibitors. those generated via solution phase methods.

Experimental Procedures Crystallization Crystals of the HDAC8:TSA complex were grown in sitting drops by Expression vapor diffusion. A 50 nl protein drop (10 mg/ml HDAC8, 25 mM Tris- The HDAC8 gene was amplified from a cDNA clone by PCR and HCl [pH 7.6], 250 mM NaCl, 0.1 mM TCEP, 2 mM TSA, and 5 mM cloned into the pSXB2 vector, a derivative of pFastbacHTa (GIBCO- CaCl2) was mixed with 50 nl of reservoir solution (12.15% [v/v] PEG Structures of Human HDAC8 1333

8000, 0.2 M calcium acetate, and 100 mM imidazole [pH 6.7]) and References incubated at 4ЊC. Crystals typically grew to their final size of 50–100 uM within 48 hr. Crystals used for data collection were soaked in Almenara, J., Rosato, R., and Grant, S. (2002). Synergistic induction reservoir solution supplemented with 30% glycerol before cryo- of mitochondrial damage and apoptosis in human leukemia cells by cooling. flavopiridol and the histone deacetylase inhibitor suberoylanilide To obtain crystals of the HDAC8:MS-344 and HDAC8:SAHA com- hydroxamic acid (SAHA). Leukemia 16, 1331–1343. plexes, HDAC8 protein (13 mg/ml HDAC8, 25 mM Tris-HCl [pH 7.6], Ayer, D.E. (1999). Histone deacetylases: transcriptional repression 100 mM NaCl, 0.1 mM TCEP, and 5 mM CaCl2) was incubated with with SIN-ers and NuRDs. Trends Cell Biol. 9, 193–198. 2 mM SAHA or MS-344. Crystals were grown from sitting drops, Bru¨ nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., where 50 nl protein was mixed with an equal volume of reservoir Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., solution (18% [v/v] PEG 8000 and 100 mM HEPES [pH 7.6]). Crystals Pannu, N.S., et al. (1998). Crystallography & NMR system: a new used for data collection were soaked in reservoir solution supple- software suite for macromolecular structure determination. Acta mented with 20% glycerol before cryocooling. Crystallogr. D. 54, 905–921. The HDAC8:CRA-A complex was crystallized from sitting drops consisting of 2 ␮l of a protein solution (12 mg/ml HDAC8, a 3-fold Buggy, J.J., Sideris, M.L., Mak, P., Lorimer, D.D., McIntosh, B., and Clark, J.M. (2000). Cloning and characterization of a novel human molar excess of the CRA-A inhibitor, 0.2 mM ZnCl2, and 1 mM EDTA) and 1 ␮l of the reservoir buffer (0.2 M KCl, 20% [w/v] PEG 3350, histone deacetylase, HDAC8. Biochem. J. 350, 199–205. and 0.1 M HEPES [pH 7.4]). Before flash cooling, crystals were David, G., Neptune, M.A., and DePinho, R.A. (2002). SUMO-1 modifi- transferred to a solution of 30% PEG 3350 and 20% gycerol. cation of histone deacetylase 1 (HDAC1) modulates its biological activities. J. Biol. Chem. 277, 23658–23663. Data Collection, Structure Determination, and Refinement Durst, K.L., Lutterbach, B., Kummalue, T., Friedman, A.D., and Hie- X-ray data were collected from cryocooled crystals at beamlines bert, S.W. (2003). The inv(16) fusion protein associates with core- 5.0.2 and 5.0.3 at the Advanced Light Source, and the data were pressors via a smooth muscle myosin heavy-chain domain. Mol. reduced using the HKL software package (Otwinowski and Minor, Cell. Biol. 23, 607–619. 1997). The structure of the HDAC8:TSA complex was solved first Finnin, M.S., Donigian, J.R., Cohen, A., Richon, V.M., Rifkind, R.A., and then used to phase the data from the other complexes. The Marks, P.A., Breslow, R., and Pavletich, N.P. (1999). Structures of positions of the two molecules in the asymmetric unit were identified a histone deacetylase homologue bound to the TSA and SAHA by molecular replacement using the program EPMR (Kissinger et inhibitors. Nature 401, 188–193. al., 1999), using the structure of HDLP from Aquifex aeolicus (Finnin et al., 1999) as a search probe. The molecular replacement led to Gao, L., Cueto, M.A., Asselbergs, F., and Atadja, P. (2002). Cloning a solution with a correlation coefficient of 29.5 and an R factor of and functional characterization of HDAC11, a novel member of the 53.3%. In spite of the poor statistics, the molecular replacement human histone deacetylase family. J. Biol. Chem. 277, 25748–25755. solution appeared to be correct based on the packing of the mole- Glaser, K.B., Staver, M.J., Waring, J.F., Stender, J., Ulrich, R.G., and cules in the unit cell and based on the appearance of poor but Davidsen, S.K. (2003). Gene expression profiling of multiple histone believable density for parts of the molecule that were not part of deacetylase (HDAC) inhibitors: defining a common gene set pro- the search model. The model was gradually improved through cycles duced by HDAC inhibition in T24 and MDA carcinoma cell lines. of manual rebuilding using Quanta (Accelrys, Inc.) and automated Mol. Cancer Ther. 2, 151–163. refinement with CNX (Bru¨ nger et al., 1998). The two molecules in Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss- the asymmetric unit were initially constrained to be identical, but PdbViewer: an environment for comparative protein modeling. Elec- this constraint was removed once most of the major manual refitting trophoresis 18, 2714–2723. was complete. Harding, M.M. (2002). Metal ligand geometry relevant to proteins After some initial refinement of the HDAC8 model, the structure and in proteins: sodium and potassium. Acta. Crystallogr. D Biol was retraced using the ARP/wARP program (warpNtrace mode) Crystallogr. 58, 872–874. (Perrakis et al., 1997), followed by further iterations of manual re- building and automated refinement. Throughout the refinement, the Hassig, C.A., Tong, J.K., Fleischer, T.C., Owa, T., Grable, P.G., Ayer, D.E., and Schreiber, S.L. (1998). A role for histone deacetylase activ- quality of the model was monitored by calculating Rfree (based on approximately 10% of the reflections) and by calculating the mo- ity in HDAC1-mediated transcriptional repression. Proc. Natl. Acad. del’s agreement with ideal geometry, using CNX and PROCHECK Sci. USA 95, 3519–3524. (Laskowski et al., 1993). Hockly, E., Richon, V.M., Woodman, B., Smith, D.L., Zhou, X., Rosa, E., Sathasivam, K., Ghazi-Noori, S., Mahal, A., Lowden, P.A., et Structure Analysis al. (2003). Suberoylanilide hydroxamic acid, a histone deacetylase Structural comparisons were carried out with Insight II (Accelrys, inhibitor, ameliorates motor deficits in a mouse model of Hunting- Inc.) and DEEPVIEW (Guex and Peitsch, 1997), and sequence com- ton’s disease. Proc. Natl. Acad. Sci. USA 100, 2041–2046. parisons with ClustalW (Thompson et al., 1994). The method de- Holm, L., and Sander, C. (1994). The FSSP database of structurally scribed by Kabsch and Sander (1983) was used to define the ele- aligned protein fold families. Nucleic Acids Res. 22, 3600–3609. ments of regular secondary structure. Figures were prepared with Showcase (SGI, Inc.) (Figure 1), Pymol (Delano Scientific, Inc.) (Fig- Hu, E., Chen, Z., Fredrickson, T., Zhu, Y., Kirkpatrick, R., Zhang, G.F., ure 4C), and with a combination of Molscript (Kraulis, 1991) and Johanson, K., Sung, C.M., Liu, R., and Winkler, J. (2000). 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Accession Numbers

The following complexes have been deposited in the Protein Data Bank: HDAC8 with MS344 (ID code 1T67), HDAC8 with SAHA (ID