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Cited by 521Histone Deacetylase Inhibitors ResearchEnzyme Structure and FunctionEnzyme function and inhibition

Histone deacetylase 6 structure and molecular basis of catalysis and inhibition

Open access
Hai, Yang, Christianson, David W·2016·Nature Chemical Biology
DOI 10.1038/nchembio.2134PMC PMC4990478
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Abstract

Histone deacetylase 6 (HDAC6) is a critical target for drug design because of its role in oncogenic transformation and cancer metastasis, and is unique among all histone deacetylases in that it contains tandem catalytic domains designated CD1 and CD2. We now report the crystal structures of CD2 from Homo sapiens HDAC6 and of CD1 and CD2 from Danio rerio HDAC6. We correlated these structures with activity measurements using 13 different substrates. The catalytic activity of CD2 from both species exhibited broad substrate specificity, whereas that of CD1 was highly specific for substrates bearing C-terminal acetyllysine residues. Crystal structures of substrate complexes yielded unprecedented snapshots of the catalytic mechanism. Additionally, crystal structures of complexes with eight different inhibitors, including belinostat and panobinostat (currently used in cancer chemotherapy), the macrocyclic tetrapeptide HC toxin, and the HDAC6-specific inhibitor N-hydroxy-4-(2-((2-hydroxyethyl)(phenyl)amino)-2-oxoethyl)benzamide, revealed surprising new insight regarding changes in Zn(2+) coordination and isozyme-specific inhibition.

Integrity signals

Registry

Crossref · 10.1038/nchembio.2134

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

7 of 15 cited sources checked · 5 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    In HDAC8, the conserved D101 accepts hydrogen bonds from the two backbone amide groups that flank the scissile acetyllysine.

    “In HDAC8, highly conserved D101 accepts hydrogen bonds from the two backbone amide groups flanking the scissile acetyllysine residue.37,38”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [37] Vannini, A. et al. Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex. EMBO Rep. 8, 879–884 (2007). source
    • cites [38] Dowling, D.P., Gantt, S.L., Gattis, S.G., Fierke, C.A. & Christianson, D.W. Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors. Biochemistry 47, 13554–13563 (2008). source

      The Results and Discussion state that strictly conserved D101 accepts hydrogen bonds from two substrate backbone NH groups. They identify those groups as belonging to the acetyllysine residue itself and the adjacent n+1 residue, rather than two residues flanking the acetyllysine. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    In HDAC8, the second histidine of the tandem pair acts as a single general base-general acid.

    “this contrasts with HDAC8, in which the second histidine of the tandem pair serves as a single general base-general acid.33”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] Gantt, S.M. et al. General base-general acid catalysis in human histone deacetylase 8. Biochemistry 55, 820–832 (2016). source
  • Findingreasoning

    K330L zCD1 has more relaxed substrate specificity than wild-type zCD1.

    “Consistent with this hypothesis, K330L zCD1 exhibited more relaxed substrate specificity in comparison with wild-type zCD1 (Supplementary Fig. 2).”
    • Also stated in: § Basis of CD1 specificity for X-K(Ac)-CO2− substrates · plain
      “K330L zCD1 exhibited more relaxed substrate specificity in comparison with wild-type zCD1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Supplementary Fig. 2

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingown data

    The 1.80 Å structure of substrate 8 bound to H574A zCD2 provides a snapshot of the tetrahedral intermediate.

    “the next snapshot of catalysis – the tetrahedral intermediate – was provided by the 1.80 Å-resolution structure of the complex between substrate 8 (derived from histone H4) and H574A zCD2.”
    Quote in the paperSubstantial claim
  • Findingown data

    In the resting zCD2 structure, the catalytic Zn2+ ion sits at the bottom of a roughly 10 Å deep tunnel and is coordinated by D612, H614, D705, and a water molecule in distorted tetrahedral geometry.

    “the crystal structure of the resting enzyme showed that the catalytic Zn2+ ion resides at the base of a ~10 Å deep tunnel, liganded by D612, H614, D705, and a water molecule with distorted tetrahedral coordination geometry.”
    Quote in the paperSubstantial claim
  • May combine two claims. 53
  • Classified as probably not a substantial claim. 9

  • May combine two claims.

    Premisecitation

    HC toxin inhibits maize HD1-B, a class I HDAC, irreversibly but inhibits HD1-A, a class II HDAC, reversibly.

    “HC toxin exhibits irreversible inhibition against maize HD1-B (a class I HDAC), but reversible inhibition against HD1-A (a class II HDAC).39”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [39] Brosch, G., Ransom, R., Lechner, T., Walton, J.D. & Loidl, P. Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum. Plant Cell 7, 1941–1950 (1995). source

      The Abstract (p. 1941) says HD1-A and HD1-B were similarly inhibited by HC toxin and describes the inhibition as reversible. That contradicts the claimed irreversible inhibition of HD1-B. The passage does not assign either enzyme to an HDAC class. (LLM, conf=0.89)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Trapoxin contains the Aoe moiety and irreversibly inhibits HDAC1 but reversibly inhibits HDAC6.

    “trapoxin contains the Aoe moiety and is a potent irreversible inhibitor of HDAC1, but it is a reversible inhibitor of HDAC6.40”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] Furumai, R. et al. Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin. Proc. Natl. Acad. Sci. USA 98, 87–92 (2001). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The quality of each model was verified with PROCHECK and MolProbity.

    “The quality of each model was verified with PROCHECK55 and MolProbity.56”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [55] Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993). source
    • cites [56] Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010). source

  • May combine two claims.

    Premisecitation

    HPOB potency against HDAC6 is comparable to SAHA-like inhibitors, but its potency against other isozymes is significantly reduced.

    “Accordingly, the potency of HPOB against HDAC6 is comparable to SAHA-like inhibitors, but HPOB potency against other isozymes is significantly reduced.43”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Lee, J.H. et al. Development of a histone deacetylase 6 inhibitor and its biological effects. Proc. Natl. Acad. Sci. USA 110, 15704–15709 (2013). source

  • May combine two claims.

    Findingown data

    With substrates 9 and 10, hCD1 in Y782F hCD12 has a catalytic efficiency of about 1,000 M−1s−1, which is robust compared with hCD2 in Y386F hCD12.

    “hCD1 (Y782F hCD12) exhibited robust catalytic efficiency (~1,000 M−1s−1) compared to hCD2 (Y386F hCD12) with substrates 9 and 10”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    zCD1 is less active than zCD2, with kcat/KM reduced 21-fold for substrate 1 and 7-fold for substrate 8.

    “zCD1 was less active than zCD2 (kcat/KM is reduced 21-fold and 7-fold for substrates 1 and 8, respectively).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The electron density map shows the scissile acetyllysine carbonyl attacked by a water molecule activated by H573 and Zn2+, forming a tetrahedral intermediate that is trapped because H574 is absent.

    “The electron density map indicated that the scissile carbonyl of acetyllysine underwent nucleophilic attack by a water molecule activated by H573 and Zn2+ to yield a tetrahedral intermediate, trapped in the active site due to the loss of the general acid H574”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Y782F hCD12, hCD1 is catalytically active with C-terminal acetyllysine substrates 9 and 10 and is strictly specific for substrates with a C-terminal acetyllysine.

    “significant hCD1 activity in Y782F hCD12 was observed with C-terminal acetyllysine peptide substrates 9 and 10, indicating that hCD1 was indeed catalytically active, but exhibited strict substrate specificity for substrates bearing C-terminal acetyllysine residues”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 1.82 Å enzyme-substrate complex shows the scissile acetyllysine carbonyl binding to the previously vacant Zn2+ site without displacing the Zn2+-bound water, giving a pentacoordinate metal ion ready for catalysis.

    “The 1.82 Å-resolution structure of the enzyme-substrate complex revealed that the scissile carbonyl of acetyllysine coordinates to the formerly vacant site on Zn2+ without displacing the Zn2+-bound water molecule, resulting in a pentacoordinate metal ion poised for catalysis”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both hCD2 and zCD2 have broad substrate specificity.

    “both hCD2 and zCD2 exhibited broad substrate specificity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the zCD2 active site, only N530 and N645 are not conserved and correspond to D567 and M682 in hCD2; these residues lie at the outer rim and far from bound inhibitors.

    “In the zCD2 active site, only N530 and N645 were not conserved, appearing as D567 and M682 in hCD2; moreover, these residues were at the outer rim of the active site and were quite distant from bound inhibitors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hCD2 structure as a maltose-binding-protein fusion resembles the zCD2 structure, with 59% sequence identity and 75% similarity and an r.m.s. deviation of 0.43 Å over 314 Cα atoms.

    “The crystal structure of hCD2 as a fusion construct with maltose binding protein was similar to that of zCD2 (59%/75% sequence identity/similarity) (Supplementary Fig. 5), with an r.m.s. deviation of 0.43 Å for 314 Cα atoms”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the tetrahedral intermediate, O1 is stabilized by Zn2+ coordination at 2.1 Å and by a hydrogen bond to Y745, while O2 is 2.6 Å from Zn2+ and hydrogen bonds to H573.

    “oxyanion atom O1 is stabilized by coordination to Zn2+ (2.1 Å) and a hydrogen bond with Y745; hydroxyl group O2 is somewhat distant from Zn2+ for inner sphere coordination (2.6 Å), but hydrogen bonds with H573.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hCD1 hydrolyzes C-terminal acetyllysine substrates only when the tandem domain assembly is intact; the isolated hCD1 domain is inactive.

    “The hydrolysis of C-terminal acetyllysine substrates by hCD1 required an intact tandem domain assembly, since hCD1 was not catalytically active as an isolated domain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The isolated hCD2 domain is about fivefold less active than hCD12, and no activity is detected for hCD1 up to 5 μM protein.

    “The individual hCD2 domain was slightly less active (5-fold) than hCD12, whereas no activity was detected with hCD1 at up to 5 μM protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    zCD1 matches zCD2 activity on substrates 9 and 10 but is less active on substrates 11-13, with kcat/KM reduced 20-380-fold.

    “zCD1 was as active as zCD2 with substrates 9 and 10, but less active than zCD2 with substrates 11–13 (20–380-fold reduced kcat/KM).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mutating hCD1 does not substantially change catalysis, but mutating hCD2 nearly eliminates catalytic activity.

    “showing that mutation of hCD1 does not significantly affect catalysis, whereas mutation of hCD2 essentially abolished catalytic activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Crystal structures were determined for human HDAC6 CD2 and for zebrafish HDAC6 CD1 and CD2.

    “We now report the crystal structures of CD2 from Homo sapiens and CD1 and CD2 from Danio rerio HDAC6”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both catalytic domains of zebrafish HDAC6 are active with fluorogenic substrates, including when each domain is assayed alone.

    “both catalytic domains of zHDAC6 were active when assayed with fluorogenic substrates, even when assayed as individual domains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In full-length human and zebrafish enzymes, CD2 accepts endo- and exo-acetyllysine substrates X-K(Ac)-X, X-K(Ac)-Pro-X, and X-K(Ac)-CO2−, while hCD1 is strictly specific for exo-acetyllysine substrates X-K(Ac)-CO2− and zCD1 is somewhat less strict.

    “in the full-length human and zebrafish enzymes, CD2 exhibited broad substrate specificity for endo- and exo-acetyllysine peptide substrates X-K(Ac)-X, X-K(Ac)-Pro-X, and X-K(Ac)-CO2−, whereas hCD1 exhibited strict substrate specificity for exo-acetyllysine peptide substrates X-K(Ac)-CO2−; substrate specificity for zCD1 was somewhat more relaxed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The gem-diol binds like the tetrahedral intermediate with substrate 8, with asymmetric Zn2+-O1 and Zn2+-O2 distances of 2.0 Å and 2.5 Å.

    “The binding of the gem-diol moiety was comparable to that observed for the binding of the tetrahedral intermediate with substrate 8, making asymmetric metal coordination interactions with Zn2+-O1 and Zn2+-O2 distances of 2.0 Å and 2.5 Å, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutating zCD2 N530 or hCD2 D567 does not significantly change activity, while mutating hCD2 S568 or zCD2 S531 lowers catalytic efficiency by 10-fold and 258-fold, respectively.

    “mutations of zCD2 N530 and hCD2 D567 did not significantly affect activity, whereas mutations of hCD2 S568 and zCD2 S531 decreased catalytic efficiency by 10-fold and 258-fold, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    H82F/F202Y zCD1 loses activity on endo-acetyllysine substrate 8 and becomes significantly specific for substrates 9 and 10, which have an exo-acetyllysine group.

    “These amino acid substitutions abolished activity with endo-acetyllysine peptide substrate 8 (Fig. 1b) and conferred significant specificity for substrates 9 and 10, which bear an exo-acetyllysine group”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The zCD1 active site is slightly more constricted, partly because K330 protrudes, hydrogen bonds to the TSA cap carbonyl, and sterically pushes it toward the L1 loop.

    “In zCD1, the active site was slightly more constricted, due in part to the protruding side chain of K330, which donates a hydrogen bond to the carbonyl group of the “cap” moiety of TSA and sterically displaces it toward the L1 loop.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HDAC6 zCD2, but not HDAC8, shows significant catalytic activity on substrates with Lys(Ac)-Pro linkages.

    “we observed significant catalytic activity for HDAC6 zCD2, but not HDAC8, using substrates with Lys(Ac)-Pro linkages”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Aoe ketone carbonyl is hydrated, so the inhibitor binds as a gem-diol(ate), similar to the trifluoroketone and the tetrahedral intermediate with substrate 8.

    “the ketone carbonyl of Aoe was hydrated so that the inhibitor binds as a gem-diol(ate), much like that observed for the trifluoroketone (Supplementary Fig. 6c) or for the tetrahedral intermediate with substrate 8 (Fig. 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    zCD1 and hCD1 have low or no activity on traditional fluorogenic substrates 1-8 but are highly specific for hydrolyzing exo-acetyllysine substrates 9 and 10, X-K(Ac)-CO2−.

    “zCD1 and hCD1 exhibited low or no activity when assayed with traditional fluorogenic substrates 1–8, but instead were very specific for the hydrolysis of exo-acetyllysine substrates 9 and 10, X-K(Ac)-CO2−”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In addition to K353, two substitutions further constrict the hCD1 active site: zCD1 F202 and H82 correspond to hCD1 Y225 and F105.

    “Two amino acid substitutions further constrict the active site of hCD1 in addition to K353 (which corresponds to K330 of zCD1): F202 and H82 in zCD1 (Fig. 4a) appear as Y225 and F105 in hCD1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    O1 hydrogen bonds with Y745, and O2 hydrogen bonds with H573 and H574.

    “O1 formed a hydrogen bond with Y745, and O2 formed a hydrogen bond with H573 and H574.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In broad-specificity inhibitor complexes, the hydroxamate OH interacts with H573 and the hydroxamate C=O interacts with Y745.

    “Additionally, the hydroxamate groups of all broad-specificity inhibitors tended toward bidentate Zn2+ coordination (Fig. 5b and Supplementary Table 5), displacing the Zn2+-bound water molecule of the resting enzyme, with the hydroxamate OH and C=O groups additionally interacting with H573 and Y745, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The L-Ala-D-Ala and L-Aoe-D-Pro linkages of HC toxin are cis, giving the cyclic tetrapeptide an unusual alternating cis-trans-cis-trans geometry.

    “the L-Ala–D-Ala and the L-Aoe–D-Pro peptide linkages of HC toxin adopted cis configurations, giving the cyclic tetrapeptide unusual alternating cis-trans-cis-trans peptide bond geometry.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In zCD2, N530 is offset so that S531 aligns with HDAC8 D101 and accepts a hydrogen bond from the acetyllysine backbone NH in substrates 1 and 8.

    “the corresponding residue in zCD2, N530, was offset such that S531 of zCD2 structurally aligned with D101 of HDAC8 and accepted a hydrogen bond from the backbone NH group of acetyllysine in substrates 1 and 8”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The corresponding residue in zCD2 is L712, which protrudes less into the active site.

    “The corresponding residue in zCD2 was L712, which does not protrude as far into the active site.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Substrate binding to zCD2 causes no major conformational changes in the L1 and L2 loops around the active site, so the active site is essentially pre-formed.

    “substrate binding to zCD2 does not trigger any major conformational changes in the L1 and L2 loops surrounding the active site, so the active site is essentially pre-formed for substrate binding”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    A trifluoroketone inhibitor binds as a tetrahedral gem-diol with Ki = 0.8 μM, mimicking the tetrahedral intermediate.

    “The tetrahedral intermediate was also mimicked by the binding of a trifluoroketone inhibitor35 as a tetrahedral gem-diol with Ki = 0.8 μM”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [35] Decroos, C., Bowman, C.M. & Christianson, D.W. Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase. Bioorg. Med. Chem. 21, 4530–4540 (2013). source

  • May combine two claims.

    Findingown data

    This residue is P519 in zCD2 and does not cause a conformational change in the corresponding tryptophan W459.

    “This residue appeared as P519 in zCD2 and did not trigger a conformational change of the corresponding tryptophan residue, W459.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Binding of HC toxin to zCD2, with Ki = 0.35 μM, causes no major conformational changes in HDAC6.

    “No major conformational changes of HDAC6 accompanied the binding of HC toxin to zCD2 (Ki = 0.35 μM)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Aoe O1 coordinates to Zn2+ at 2.0 Å and hydrogen bonds with Y745; O2 weakly coordinates to Zn2+ at 2.5 Å and hydrogen bonds with H573 and H574.

    “the Aoe O1 atom coordinates to Zn2+ (2.0 Å) and hydrogen bonds with Y745; the O2 atom makes a weak coordination interaction with Zn2+ (2.5 Å), and hydrogen bonds with H573 and H574.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hydroxamate group of HPOB coordinates to Zn2+ only through its OH group and does not displace the Zn2+-bound water molecule.

    “In contrast, the hydroxamate group of the HDAC6-selective inhibitor HPOB (Ki = 1.0 nM) coordinated to Zn2+ only through its OH group, and it did not displace the Zn2+-bound water molecule; the Zn2+ ion is pentacoordinate with square pyramidal geometry (Fig. 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The product acetate anion binds with similar interactions, with Zn2+-O1 and Zn2+-O2 distances of 2.2 Å and 2.1 Å.

    “similar interactions were observed for the binding of the product acetate anion with Zn2+-O1 and Zn2+-O2 distances of 2.2 Å and 2.1 Å, respectively”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The Aoe backbone NH hydrogen bonds to S531, and water-mediated hydrogen bond networks connect the Aoe backbone carbonyl to H614 and the HC toxin L-Ala backbone NH to H463.

    “The backbone NH group of Aoe donated a hydrogen bond to S531, and water-mediated hydrogen bond networks were observed between the backbone carbonyl of Aoe and H614, and the backbone NH group of HC toxin L-Ala and H463”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Y225F/Y782F hCD12 has nearly 100-fold higher activity on endo-acetyllysine fluorogenic substrates than hCD12 Y782F, while K353L/Y782F hCD12 has a more modest 10-fold increase.

    “Y225F/Y782F hCD12 exhibited nearly 100-fold enhanced activity with endo-acetyllysine fluorogenic substrates when compared to hCD12 Y782F, whereas K353L/Y782F hCD12 exhibited a more modest 10-fold activity enhancement”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The CD2 domains of human and zebrafish HDAC6 can accommodate proline at the +1 position relative to the scissile acetyllysine residue, whereas HDAC8 cannot because of the hydrogen bond between D101 and the backbone NH of the +1 residue.

    “The CD2 domains of human and zebrafish HDAC6 can accommodate proline at the +1 position relative to the scissile acetyllysine residue, whereas HDAC8 cannot based on the hydrogen bond between D101 and the backbone NH of the +1 residue.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    H573 may act as a general base helping Zn2+ activate the nucleophilic water, so the two histidines in the zCD2 active site may have separate general base and general acid roles.

    “H573 could serve as a general base to assist Zn2+ in activating the nucleophilic water molecule, so the tandem histidines in the HDAC6 zCD2 active site may serve separate general base-general acid functions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    HDAC6 removes the acetyl group from K40 of α-tubulin and thereby controls microtubule dynamics.

    “HDAC6 catalyzes the deacetylation of K40 in the α-tubulin subunit of the microtubule,24 thereby regulating microtubule dynamics”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002). source

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    HDAC6 is an important drug target because it contributes to oncogenic transformation and cancer metastasis.

    “Histone deacetylase 6 (HDAC6) is a critical target for drug design due to its role in oncogenic transformation and cancer metastasis”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Blocking HDAC6 increases α-tubulin acetylation and suppresses microtubule dynamics, whereas having more HDAC6 lowers tubulin acetylation and raises cell motility.

    “HDAC6 inhibition leads to hyperacetylation of α-tubulin and suppression of microtubule dynamics, while overexpression of HDAC6 reduces tubulin acetylation levels and increases cell motility.25”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Szyk, A. et al. Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell 157, 1405–1415 (2014). source

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationreasoning

    K330 may act as a gatekeeper that gives specificity for C-terminal acetyllysine substrates by hydrogen bonding to the acetyllysine α-carboxylate.

    “we hypothesized that K330 is a “gatekeeper” that confers specificity toward C-terminal acetyllysine substrates by hydrogen bonding with the α-carboxylate group of acetyllysine.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Substrate 8 probably stays bound as a metastable tetrahedral intermediate because enzymes generally bind transition-state-like structures more favorably than substrate-like structures.

    “Substrate 8 likely remained bound as a metastable tetrahedral intermediate because an enzyme generally favors the binding of a transition state-like structure over the binding of a substrate-like structure”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted). May combine two claims.

    Interpretationown data

    Y202's hydroxyl may hydrogen bond to the acetyllysine α-carboxylate to prevent endo-acetyllysine binding, so that K330 and Y202, corresponding to hCD1 K353 and Y225, enforce specificity for exo-acetyllysine substrates X-K(Ac)-CO2−.

    “We hypothesized that the hydroxyl group of Y202 hydrogen bonds with the α-carboxylate group of the acetyllysine substrates to abolish the binding of endo-acetylysine substrates, such that both K330 and Y202 (K353 and Y225 in hCD1) enforce specificity for substrates bearing an exo-acetyllysine group, X-K(Ac)-CO2−”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    In H574A zCD2, the substrate carbonyl is activated enough for nucleophilic attack, but the tetrahedral intermediate cannot collapse because H574 is missing to protonate the leaving amino group.

    “the substrate carbonyl was sufficiently activated to undergo nucleophilic attack, but the resulting tetrahedral intermediate was locked in place, deep in the active site pocket – it could not collapse without general acid H574 to protonate the leaving amino group.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationreasoning

    The L-Aoe-D-Pro-L-Ala fragment of HC toxin may partially mimic a substrate site in a reverse turn, such as X-Lys(Ac)-Pro-X, or other proline-containing loops or cis-Lys(Ac)-X segments.

    “we hypothesize that the L-Aoe-D-Pro-L-Ala fragment of HC toxin partially mimics a potential substrate site located in a reverse turn, such as X-Lys(Ac)-Pro-X, as well as other proline-containing loop structures or segments containing cis-Lys(Ac)-X linkages.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The stronger O1-Zn2+ interaction may mean O1 is ionized to an oxyanion, which would also be stabilized inductively by the nearby trifluoromethyl group.

    “The stronger interaction of O1 with Zn2+ may indicate ionization to an oxyanion, which would also be inductively stabilized by the adjacent trifluoromethyl group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The amide bond after the scissile acetyllysine is not required for substrate binding to zCD2 and hCD2 because it makes no direct contacts with active site residues.

    “we suggest that the amide bond following the scissile acetyllysine residue is dispensable for substrate binding to zCD2 and hCD2, since it makes no direct interactions with active site residues.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The backbone amide group after acetyllysine hydrogen bonds only to water, suggesting that any amino acid, including proline, can occupy that position.

    “The backbone amide group following acetyllysine hydrogen bonded only with a water molecule, suggesting that any amino acid – including proline – could be accommodated at this position.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Comparison of different zCD2-inhibitor structures highlights the structural basis for HDAC6-specific inhibitor design, which exploits a previously unobserved Zn2+ coordination mode by the hydroxamate group of HPOB: inhibitor binding does not require displacement of Zn2+-bound water.

    “Finally, comparison of different zCD2-inhibitor structures highlights the structural basis for HDAC6-specific inhibitor design, which exploits a previously unobserved Zn2+ coordination mode by the hydroxamate group of HPOB: inhibitor binding does not require displacement of Zn2+-bound water.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The inhibitor binding modes observed in zebrafish CD2 crystals are representative of human HDAC6.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No statistical methods were used to predetermine sample size.

    “No statistical methods were used to predetermine sample size.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Zinc hydrolases activate a substrate carbonyl group for direct nucleophilic attack by a water molecule using Zn2+ coordination and a hydrogen bond interaction to polarize the carbonyl group; the nucleophilic water molecule is activated by Zn2+ coordination and a general base.

    “In general, zinc hydrolases activate a substrate carbonyl group for direct nucleophilic attack by a water molecule using Zn2+ coordination and a hydrogen bond interaction to polarize the carbonyl group; the nucleophilic water molecule is activated by Zn2+ coordination and a general base.44,45”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [44] Matthews, B.W. Structural basis of the action of thermolysin and related zinc peptidases. Acc. Chem. Res. 21, 333–340 (1988). source
    • cites [45] Christianson, D.W. & Lipscomb, W.N. Carboxypeptidase A. Acc. Chem. Res. 22, 62–69 (1989). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Assays were performed in triplicate at room temperature.

    “Assays were performed in triplicate at room temperature.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    No backbone torsion angles adopt disallowed conformations in any structure.

    “No backbone torsion angles adopt disallowed conformations in any structure.”
    Quote in the paperNot a substantial claim
60 of 60

  • The paper relies on this without support.

    Before this study, no crystal structures of HDAC6 catalytic domains existed.

    “Despite these unresolved structure-function relationships, no crystal structures of HDAC6 catalytic domains have been available until now.”

  • The paper relies on this without support.

    The active site of a Zn2+ hydrolase is adapted to allow water to attack almost any carbonyl group and to stabilize the resulting tetrahedral species.

    “The active site of a Zn2+ hydrolase has evolved to enable nucleophilic attack of water at virtually any carbonyl group, preferentially stabilizing the resulting tetrahedral species.”

  • The paper relies on this without support.

    The inhibitor binding modes observed in zebrafish CD2 crystals are representative of human HDAC6.

    “Finally, comparison of different zCD2-inhibitor structures highlights the structural basis for HDAC6-specific inhibitor design, which exploits a previously unobserved Zn2+ coordination mode by the hydroxamate group of HPOB: inhibitor binding does not require displacement of Zn2+-bound water.”

  • The paper relies on this without support.

    The Aoe side chain carbonyl is isosteric with the scissile carbonyl of acetyllysine.

    “Since the carbonyl group of the Aoe side chain is isosteric with the scissile carbonyl of acetyllysine, the binding of Aoe-containing peptides to any HDAC should enable facile hydration of the Aoe ketone carbonyl, as observed in the zCD2-HC toxin complex.”

  • The paper relies on this without support.

    The active site geometry of class I HDACs is sufficiently similar to zCD2 that the nucleophile and inhibitor binding conclusions transfer.

    “Presumably, inhibitor binding to class I HDAC active sites allows for a closer contact between the nucleophilic thiol and the electrophilic epoxide to enable covalent bond formation.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Recent work on HDAC6/inhibitor complexes demonstrates that L1 loop conformational stability is important for interactions of L1 loop residues with inhibitor capping groups to achieve HDAC6 selectivity inhibition.

    “the recent work on HDAC6/inhibitor complexes demonstrate that L1 loop conformational stability is important for the interactions of L1 loop residues with inhibitors capping groups to achieve HDAC6 selectivity inhibition 42-46”

    From Characterization of Histone Deacetylase 8 (HDAC8) Selective Inhibition Reveals Specific Active Site Structural and Functional Determinants (2018) · cites this paper as [42]

    The Results section (‘HDAC6 inhibitor selectivity’; page unavailable) reports that inhibitor caps contact L1-loop residues, but identifies those inhibitors as broad-specificity. The Discussion (page unavailable) describes the L1 loop as relatively rigid, while attributing the structural basis for HDAC6-selective inhibition to HPOB’s distinct zinc-coordination mode. It does not establish that L1-loop rigidity enables cap interactions to achieve selectivity. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Selective inhibition appears to rely on small structural differences between HDAC1-3 and HDAC6 and on small thermodynamic differences in selective inhibitor binding.

    “Selective inhibition appears to rely on small structural differences between HDAC1-3 and HDAC6 and on small thermodynamic differences in selective inhibitor binding 42-46 .”

    From Characterization of Histone Deacetylase 8 (HDAC8) Selective Inhibition Reveals Specific Active Site Structural and Functional Determinants (2018) · cites this paper as [42]

    The Results section on HDAC6 inhibitor selectivity describes structural differences in how broad-specificity inhibitors and the HDAC6-selective inhibitor HPOB bind to HDAC6. It does not establish that selectivity relies on small structural differences between HDAC1–3 and HDAC6, or on small thermodynamic differences in inhibitor binding. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • HDAC6 catalytic domain CD1 has a conserved active site lysine gatekeeper that dictates specificity for carboxy-terminal acetyllysine substrates bearing a negatively charged α-carboxylate group.

    “We recently reported the X-ray crystal structures of both catalytic domains (CDs) of HDAC6, showing that a conserved active site lysine serves as a gatekeeper in CD1, in that it dictates specificity for carboxy-terminal acetyllysine substrates bearing a negatively charged α-carboxylate group17.”

    From Histone deacetylase 10 structure and molecular function as a polyamine deacetylase (2017) · cites this paper as [17]

  • HDAC10 is not an efficient lysine deacetylase compared with HDAC6.

    “In comparison with its closest relative, the cytosolic isozyme HDAC6 (ref. 17), HDAC10 was clearly not an efficient lysine deacetylase.”

    From Histone deacetylase 10 structure and molecular function as a polyamine deacetylase (2017) · cites this paper as [17]

  • D94 corresponds to acetyllysine binding residues D101 of hHDAC8 and S568 of hHDAC6 CD2.

    “Interestingly, D94 corresponds to acetyllysine binding residues D101 of hHDAC8 and S568 of hHDAC6 CD2 (refs 17, 30, 31).”

    From Histone deacetylase 10 structure and molecular function as a polyamine deacetylase (2017) · cites this paper as [17]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Substrate 1 complex is assigned to two different mutants

    Minor

    The crystallization method identifies the zCD2-substrate 1 complex as Y745F, but the structure-determination section repeatedly identifies it as Y785F. These are different residue numbers for the same reported complex.

1 of 1

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Authors
Hai, Y., & Christianson, D. W.
Affiliations
  • University of Pennsylvania (US)
  • Harvard University (US)
Corresponding author
Christianson, David W
ORCID
Venue
Nature Chemical Biology
Publisher
Springer Science and Business Media LLC
Published
2016-07-25
Volume
12
Issue
9
Pages
741-747
ISSN
  • 1552-4450 (ISSN-L)
  • 1552-4469
DOI
10.1038/nchembio.2134
PMC
PMC4990478
OpenAlex
W2499955277
Cited by
521
References
61
Citations by year
2026: 56 · 2025: 76 · 2024: 50 · 2023: 55 · 2022: 47 · 2021: 57 · 2020: 54 · 2019: 46 · 2018: 42 · 2017: 29 · 2016: 9
FWCI
23.03
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Chemical Biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of General Medical Sciences (R01GM049758)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Histone Deacetylase Inhibitors Research
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme function and inhibition
Keywords
HDAC6, crystal structure, substrate specificity, catalytic mechanism, belinostat, panobinostat, HC-toxin, zinc coordination, small molecule inhibitors, cancer drug targets
MeSH terms
Animals, Histone Deacetylases, Humans, Models, Molecular, Structure-Activity Relationship, Substrate Specificity, Zebrafish, Molecular Structure, Biocatalysis, Histone Deacetylase Inhibitors

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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