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Letter
Binding of the Microbial Cyclic Tetrapeptide Trapoxin
A to the Class I Histone Deacetylase HDAC8
Nicholas J. Porter, and David W. Christianson
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00330 • Publication Date (Web): 28 Aug 2017
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10 Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I
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12 Histone Deacetylase HDAC8
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21 Nicholas J. Porter and David W. Christianson*
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Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania,
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35 Philadelphia, PA 19104-6323, United States
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46 *author to whom correspondence should be sent: E-mail chris@sas.upenn.edu
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6 Abstract
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8 Trapoxin A is a microbial cyclic tetrapeptide that is an essentially irreversible inhibitor of
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10 class I histone deacetylases (HDACs). The inhibitory warhead is the α,β-epoxyketone side-
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chain of (2S,9S)-2-amino-8-oxo-9,10-epoxydecanoic acid (L-Aoe), which mimics the side-chain
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15 of the HDAC substrate acetyl-L-lysine. We now report the crystal structure of the HDAC8–
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17 trapoxin A complex at 1.24 Å resolution, revealing that the ketone moiety of L-Aoe undergoes
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19 nucleophilic attack to form a zinc-bound tetrahedral gem-diolate that mimics the tetrahedral
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21 intermediate and its flanking transition states in catalysis. Mass spectrometry, activity
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23 measurements, and isothermal titration calorimetry confirm that trapoxin A binds tightly (Kd = 3 ±
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1 nM) and does not covalently modify the enzyme, so the epoxide moiety of L-Aoe remains
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28 intact. Comparison of the HDAC8–trapoxin A complex with the HDAC6-HC toxin complex
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30 provides new insight regarding the inhibitory potency of L-Aoe-containing natural products
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32 against class I and class II HDACs.
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3 Ever since the discovery of histone acetylation in transcriptional regulation more than 50
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6 years ago,1 thousands of histone and non-histone proteins have been identified as lysine
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8 acetylation targets in the mammalian acetylome.2-4 The reversible acetylation of L-lysine side
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10 chains is a critical molecular strategy for the regulation of protein function in vivo and rivals
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12 phosphorylation in the regulation of diverse biological processes, including the cell cycle and
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14 central carbon metabolism.5,6 The acetylation of a specific L-lysine residue on a target protein is
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16 catalyzed by a histone acetyl transferase (HAT) using acetyl CoA as a co-substrate;7,8 the
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19 hydrolysis of acetyl-L-lysine to form free L-lysine and acetate is catalyzed by a histone
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21 deacetylase (HDAC).9,10
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23 Upregulated HDAC activity is associated with tumorigenesis, neurodegenerative
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25 diseases, and immune disorders, so this enzyme family represents an attractive target for
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27 therapeutic intervention.11,12 Phylogenetic analysis indicates four classes of deacetylases: class
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29 I HDACs (1, 2, 3, and 8), class IIa (4, 5, 7, and 9) and class IIb (6 and 10) HDACs, class III
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32 HDACs (sirtuins 1-7), and the single class IV enzyme HDAC11.13 Class I, II, and IV HDACs
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34 require Zn2+ or Fe2+ for optimal catalytic activity.14 The first crystal structure of a metal-
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36 dependent deacetylase15 revealed an α/β fold identical to that first observed in arginase.16,17
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38 This fold is distinct from that adopted by sirtuins, which employ a different catalytic mechanism
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40 that requires the cofactor NAD+.18 Among the metal-dependent HDACs, HDAC8 was the first to
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yield a crystal structure.19,20 Important catalytic residues in the HDAC8 active site include
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45 tandem histidines (electrostatic catalyst H142 and general base-general acid H143)21,22 and a
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47 conformationally-flexible23 tyrosine residue that assists the Zn2+ ion in the polarization of the
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49 substrate carbonyl group.24,25
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51 The tightest-binding HDAC inhibitors are those capable of coordinating to the active site
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53 Zn2+ ion and also forming hydrogen bonds with catalytically-important active site residues.
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56 Currently, four HDAC inhibitors are approved for clinical use in cancer chemotherapy, one of
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58 which is the cyclic depsipeptide romidepsin.26,27 Macrocyclic HDAC inhibitors such as
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3 romidepsin or the related depsipeptide largazole28,29 comprise rigid scaffolds to which a Zn2+-
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6 coordinating functional group is attached through a linker that is the approximate length of a
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8 lysine side chain.
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10 Trapoxin A, first isolated from the microbial parasite Helicoma ambiens, is a macrocyclic
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12 tetrapeptide HDAC inhibitor with the amino acid sequence cyclo-[L-Phe–L-Phe–D-hPro–L-Aoe]
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14 (hPro = homoproline, also known as pipecolic acid; L-Aoe = (2S,9S)-2-amino-8-oxo-9,10-
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epoxydecanoic acid).30 The α,β-epoxyketone moiety of the L-Aoe side chain serves as the Zn2+-
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19 coordinating group and its ketone carbonyl group is isosteric with the scissile carbonyl of the
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21 HDAC substrate acetyl-L-lysine (Figure 1). Trapoxin A is proposed to act as an irreversible
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23 inhibitor of class I HDACs31,32 and was used for the first isolation of HDAC1 from the nuclear
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25 extracts of human Jurkat T cells.33 The epoxide moiety of the L-Aoe side chain was thought to
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27 react with the nucleophilic side chain of an active site residue; however, a covalent enzyme-
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inhibitor complex was not observable by SDS-PAGE/autoradiography.32 Curiously, although
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32 trapoxin A was found to irreversibly inhibit HDAC1, a class I enzyme, it was found to reversibly
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34 inhibit the class II enzyme HDAC6.34 The molecular basis of these activity differences has
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36 remained unclear in the absence of structural data.
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38 Here, we report the first X-ray crystal structure of trapoxin A complexed with a class I
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40 histone deacetylase, HDAC8, at 1.24 Å resolution (Figure 2a). Experimental procedures,
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including the preparation of a new HDAC8 construct for crystallographic studies, are outlined in
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45 the Supporting Information (data collection and refinement statistics are recorded in Supporting
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47 Information Table 1). The structure of the HDAC8–trapoxin A complex reveals that the
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49 conformation of the tetrapeptide backbone of trapoxin A is identical to that observed in the
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51 crystal structure of the uncomplexed inhibitor,30 with root-mean-square (rms) deviations of 0.21–
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53 0.24 Å for 16 main chain atoms of the cyclic peptide backbone; the L-Aoe side chains adopt
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56 different conformations (Supporting Information Figure S1). All peptide linkages of trapoxin A
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58 adopt the trans configuration except for the Phe-hPro linkage, which forms a cis-peptide. All
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3 three backbone NH groups donate hydrogen bonds to the side-chain carboxylate group of
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6 D101. The hydrogen bonds between D101 and the backbone NH groups of L-Aoe and the
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8 adjacent L-Phe residue are similar to those observed between D101 and linear tetrapeptide
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10 substrates bound to inactivated HDAC8 (Supporting Information Figure S2).24,25 Apart from
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12 interactions described below for the L-Aoe side chain, no other direct enzyme-inhibitor hydrogen
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14 bonds are observed. Although there are no intramolecular hydrogen bonds in trapoxin A, the
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16 side chains of its two L-Phe residues make a favorable quadrupole-quadrupole interaction with
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19 edge-to-face geometry.
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21 Conformational changes are required in the enzyme active site to accommodate the
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23 steric bulk of the rigid peptide macrocycle in comparison with substrate binding, primarily in the
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25 L2 loop (residues G86-I108, of which D87-I94 are disordered). Relative to the structure of
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27 H143A HDAC8 complexed with a tetrapeptide substrate,25 the greatest change is observed for
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Y100, which undergoes a 116˚ change in side chain torsion angle χ1 (Supporting Information
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32 Figure S2). Similar conformational flexibility of Y100 accommodates the binding of the
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34 macrocyclic depsipeptide inhibitor largazole.29 In the HDAC8–trapoxin A complex, the
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36 conformational change of Y100 is triggered by one of the inhibitor L-Phe residues, with which
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38 one of two observed conformers makes a favorable edge-to-face interaction.
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40 Surprisingly, the ketone carbonyl of the L-Aoe side chain undergoes nucleophilic attack
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by water upon binding to HDAC8, such that the inhibitory species is a gem-diolate (or perhaps a
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45 gem-diol) stabilized by Zn2+ coordination and three hydrogen bonds. Thus, trapoxin A mimics
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47 the binding of the tetrahedral intermediate and its flanking transition states in catalysis; the
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49 origins of high affinity are undoubtedly rooted in the fact that trapoxin A binds as an analogue of
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51 the postulated transition state. The Zn2+–O1 and Zn2+–O2 distances for the gem-diolate are 2.5
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53 Å and 1.9 Å, respectively. The O1 hydroxyl group also forms hydrogen bonds with H142 and
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56 H143 (O–O separations = 2.7 Å each), and the O2 oxyanion accepts a hydrogen bond from
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58 Y306 (O–O separation = 2.6 Å). While it is unusual to see an unactivated ketone binding as a
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3 tetrahedral gem-diolate, which exists to less than 0.2% in aqueous solution (based on the
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6 hydration of the unactivated ketone carbonyl of acetone in aqueous solution35), it is notable that
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8 the L-Aoe side chain of the cyclic tetrapeptide inhibitor HC toxin (Figure 1) similarly undergoes
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10 nucleophilic attack in the recently-determined structure of its complex with catalytic domain 2 of
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12 HDAC6.36 This behavior is also reminiscent of the binding of unactivated aldehyde and ketone
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14 substrate analogues to carboxypeptidase A, which similarly bind as tetrahedral gem-diolate
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16 transition state analogues.37,38
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19 In the HDAC8–trapoxin A complex, the epoxide ring of the L-Aoe side chain is clearly
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21 intact and makes no hydrogen bond interactions with any enzyme residues or water molecules
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23 (Figure 2b). The closest side chains to the epoxide moiety are those of W141, H142, C153, and
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25 Y306 with interatomic separations of 3.3–3.7 Å. The epoxide moiety is believed to be required
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27 for essentially irreversible inhibitory activity against class I HDACs, based on the lack of
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29 irreversible inhibitory activity for the cyclic tetrapeptide inhibitor apicidin (Figure 1), which lacks
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32 an epoxide moiety.30 Thus, it is curious that the epoxide moiety of trapoxin A does not react with
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34 the enzyme. However, the crystal structure reveals that although the epoxide moiety binds in
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36 the vicinity of catalytic general base H143 and highly conserved C153, neither of these potential
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38 nucleophiles is positioned or oriented for nucleophilic attack at the epoxide (Figure 2b).
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41 We confirmed the irreversibility of trapoxin A inhibition by assaying HDAC8 activity
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43 following multiple rounds of dialysis of the enzyme-inhibitor complex. Regeneration of activity
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45 was not observed for HDAC8 preincubated with a 10-fold molar excess of trapoxin A, but was
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47 observed under the same conditions using apicidin (Figure 3). This result confirms that the
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epoxide moiety is required for essentially irreversible inhibition. However, this result does not
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52 prove that a covalent enzyme-inhibitor complex is formed.
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54 To study the covalent modification of HDAC8 by trapoxin A in solution, we employed
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3 hours with 10 molar equivalents of trapoxin A and subsequently digested with trypsin. Prior to
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6 digestion, mass peaks corresponding to HDAC8 covalently modified with one or two trapoxin A
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8 molecules were observed for this sample by MALDI mass spectrometry (Supporting Information
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10 Figure S3). Following trypsin digestion, LC-MS/MS analysis, and sequence analysis for residue
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12 modifications, a mass shift corresponding to the molecular weight of trapoxin A was sporadically
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14 observed for various cysteine and histidine residues located on the surface of HDAC8
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16 (Supporting Information Figure S4 and Table S2). Only nonspecific labeling of the enzyme was
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19 observed in the presence of excess inhibitor, with no particular preference for covalent
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21 modification in the active site. Additionally, incubation of trapoxin A for 1 hour in the presence
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23 and absence of HDAC8 followed by LC-MS analysis indicated the presence of only intact
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25 trapoxin A (i.e., with an intact epoxide ring; Supporting Information Figure S5). These results
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27 strongly suggest that trapoxin A is simply an exceptionally tight-binding, noncovalent transition
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29 state analogue inhibitor of HDAC8. As noted by Schramm and colleagues,39 transition state
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32 analogues with essentially irreversible binding behavior are feasible for enzymes that exhibit
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34 catalytic rate enhancements of 1010 or greater, which is likely the case for an amide hydrolase
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36 such as HDAC8 based on uncatalyzed amide bond hydrolysis half-lives measured in centuries
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38 by Radzicka and Wolfenden.40
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40 Isothermal titration calorimetry (ITC) measurements yield HDAC8 dissociation constants
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Kd = 3 ± 1 nM for trapoxin A and Kd = 250 ± 70 nM for apicidin (Supporting Information Figure
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45 S6), indicating 83-fold enhanced binding affinity for trapoxin A relative to apicidin. Given the
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47 structural similarity between the two cyclic tetrapeptides, and the fact that the macrocycle
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49 makes very few intermolecular interactions in the HDAC8–trapoxin A complex, it is clear that the
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51 epoxide moiety of trapoxin A is indeed responsible for tight binding to HDAC8.
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53 ITC measurements of HDAC8 active site variants indicate that H142 is critical for
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trapoxin A binding just as it is important for catalysis, since the H142A mutation results in
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58 significantly weaker affinity with Kd = 17 ± 5 µM – H142A HDAC8 exhibits a 233-fold reduced
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3 kcat/KM value relative to wild-type HDAC8.22 This residue functions in catalysis with a positively
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6 charged imidazolium group that serves as an electrostatic catalyst.22 Electron density is
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8 observed for the Nε-H proton of H142 in the 1.24 Å electron density map (Supporting
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10 Information Figure S7), so the H142 imidazolium group must similarly stabilize the zinc-bound
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12 gem-diolate.
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14 Although the L-Aoe side chains of trapoxin A and HC toxin bind to HDAC8 and HDAC6,
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16 respectively, as the gem-diolate, there are notable differences between the structures of each
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19 enzyme-inhibitor complex that may explain the tighter binding of these inhibitors to class I
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21 HDACs.34 In the class IIb HDAC6-HC toxin complex (Ki = 350 nM),36 the C–O bond of the
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23 epoxide adopts an energetically unfavorable eclipsed conformation with respect to the C–O
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25 bond of the hydroxyl group of the zinc-bound gem-diolate (Figure 4a); in the class I HDAC8–
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27 trapoxin A complex (Kd = 3 nM), the C–O bond of the epoxide adopts an energetically favorable
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29 staggered conformation (Figure 4b). The energetically unfavorable eclipsed conformation in the
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32 HDAC6-HC toxin complex appears to be caused by the bulky P571 residue in the L3 loop,
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34 conserved in all class II HDACs – if the epoxide adopted an energetically favorable staggered
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36 conformation in the HDAC6 active site, the epoxide methylene group would clash with P571. In
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38 class I HDACs, P571 is not conserved and the active site is more open. Thus, a more favorable
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40 binding conformation is accessible to the epoxyketone moiety only in the active site of a class I
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HDAC.
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45 Although the α,β-epoxyketone epoxide moiety of trapoxin A remains intact in the crystal
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47 structure of its complex with HDAC8, it is notable that this novel functionality is chemically
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49 reactive in the binding of inhibitors to other enzymes. For example, the proteasome inhibitor
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51 carfilzomib contains an α,β-epoxyketone that forms a covalent adduct to block proteasome
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function.41 The crystal structure of a similar natural product, epoxomicin, complexed with the
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56 yeast 20S proteasome indicates a multistep cyclization sequence leading to the formation of a
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morpholino ring between the former α,β-epoxyketone of the inhibitor and the reactive N-terminal
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6 threonine residue of the proteasome subunit.42 A two-step mechanism for inhibitor binding is
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8 initiated by nucleophilic attack of the threonine hydroxyl group at the epoxyketone carbonyl
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10 followed by a 6 Exo-Tet ring closure reaction between the α-amino group of threonine and the
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12 epoxide to generate the morpholino product.43 The chemistry of inhibitor binding in this system
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15 indicates that the carbonyl group appears to be more reactive than the epoxide of the α,β-
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17 epoxyketone moiety. This is consistent with reactivity trends observed in organic synthesis for
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19 various α,β-epoxycarbonyl derivatives, where the carbonyl group preferentially undergoes
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21 nucleophilic addition while leaving the epoxide moiety intact.44-46 With respect to trapoxin A, it is
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notable that an additional peak is observed in mass spectra consistent with gem-diol formation
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26 even in the absence of enzyme (Supporting Information Figure S5).
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28 Finally, it is interesting to compare the structures of zinc coordination polyhedra in
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30 different HDAC8-inhibitor complexes (Supporting Information Figure S8). Only one oxygen of
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32 the gem-diolate of trapoxin A is sufficiently close for inner-sphere coordination, so the overall
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34 zinc coordination geometry is best described as 4-coordinate distorted tetrahedral. In this
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regard, zinc coordination geometry approaches that observed in the HDAC8–largazole complex,
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39 which exhibits nearly perfect tetrahedral coordination geometry through the binding of the
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41 largazole thiolate group.29 In contrast with these examples, the hydroxamate group of
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43 trichostatin A coordinates to zinc in bidentate fashion, so that the overall coordination geometry
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45 is 5-coordinate square pyramidal.20
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47 In conclusion, the current work demonstrates that trapoxin A is an essentially irreversible
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50 noncovalent inhibitor of HDAC8: the α,β-epoxyketone side chain of the inhibitor undergoes
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52 nucleophilic attack by zinc-bound water to bind as a tetrahedral gem-diolate transition state
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54 analogue. Along with a favorable staggered conformation of the intact epoxide moiety relative to
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3 the zinc-bound gem-diolate, these structural features contribute to an exceptionally tight
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6 enzyme-inhibitor complex effectively locked into the enzyme active site.
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10 ASSOCIATED CONTENT
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12 Supporting Information
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14 The Supporting Information is available free of charge on the ACS Publications website at DOI:
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16 Detailed methods, figures, and tables
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19 Accession Code
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21 The atomic coordinates and crystallographic structure factors of the HDAC8-trapoxin A complex
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23 have been deposited in the Protein Data Bank (www.rcsb.org) with accession code 5VI6.
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27 AUTHOR INFORMATION
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29 Corresponding Author
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32 *Tel.: 215-898-5714. E-mail: chris@sas.upenn.edu
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34 ORCHID
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36 David W. Christianson: 0000-0002-0194-5212
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38 Funding
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40 We thank the National Institutes of Health for grant GM49758 in support of this research.
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42 Notes
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45 The authors declare no competing financial interest.
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49 ACKNOWLEDGMENT
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51 N.J.P. thanks the Department of Chemistry at the University of Pennsylvania for the award of a
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53 graduate research fellowship. Additionally, we thank C. Smith at beamline 9-2 of the Stanford
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Synchrotron Radiation Lightsource (SSRL) for assistance with data collection. The SSRL, SLAC
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58 National Accelerator Laboratory, is supported by the DOE Office of Science, Office of Basic
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3 Energy Sciences, under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular
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6 Biology Program is supported by the DOE Office of Biological and Environmental Research, and
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8 by the NIH, National Institute of General Medical Sciences (including P41GM103393).
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42 Figure 1. The canonical HDAC substrate peptidyl acetyl-L-lysine and structurally-related cyclic
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44 tetrapeptide inhibitors. The ketone carbonyl group of each inhibitor is isosteric with the scissile
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37 Figure 2. 1.24 Å-resolution structure of the HDAC8–trapoxin A complex. (a) Stereoview of
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39 trapoxin A (C = orange, N = blue, O = red) bound in the active site of HDAC8 (C = light blue, N =
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41 blue, O = red). The simulated annealing omit map of trapoxin A (green) is contoured at 3.0σ.
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43 Metal coordination and hydrogen bond interactions are indicated by solid and dashed black
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lines, respectively. (b) Close-up stereoview showing the zinc-bound gem-diolate. Atomic color
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Figure 3. HDAC activity assays. Recovered HDAC8 activity from samples incubated with
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40 DMSO, apicidin, and trapoxin A (TpxA) after no (gray), one (orange) or two (blue) rounds of
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31 Figure 4. Epoxyketone binding modes. (a) HC toxin (C = yellow, N = blue, O = red) bound to
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