Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase Stefanie Kern1, Daniel Riester2, Christian Hildmann2, Andreas Schwienhorst2 and Franz-Josef Meyer-Almes1 Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut fur Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany ¨ Keywords FB188; HDAH; histone deacetylase; protein denaturation; protein stablization Correspondence F.-J Meyer-Almes, Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, Schnittspahnstr 12, 64287 Darmstadt, Germany Fax: + 1649 6151168404 Tel: + 1649 6151168406 E-mail: meyer-almes@h-da.de Website: http://www.h-da.de/cub/ (Received 24 January 2007, revised 11 May 2007, accepted 15 May 2007) doi:10.1111/j.1742-4658.2007.05887.x Histone deacetylases are major regulators of eukaryotic gene expression Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures The refolding kinetics has shown to be strongly dependent on Zn2+ and to a lesser extent on K+, which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJỈmol)1 upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively Nucleosomal histones are subject to a variety of post-transcriptional covalent modifications, including acetylation, methylation, phosphorylation and ubiquitination [1] Reversible histone acetylation has been shown to facilitate access of the transcriptional machinery to DNA by disruption of nucleosome–nucleosome and nucleosome–DNA interactions [2–4] Acetylation of histone proteins occurs at the e-amino group of lysine residues near the N-termini of these proteins The steady-state histone acetylation level is the result of opposing actions of histone acetyltransferases and histone deacetylases (HDACs) In particular, HDACs are promising therapeutic targets on account of their involvement in regulating genes involved in cell cycle Abbreviations CypX, cyclopentylpropionyl hydroxamate; DGu, free energy of unfolding; Gdn-HCl, guanidine hydrochloride; HDAC, histone deacetylase; HDAH, histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188; meq, equilibrium parameter which reflects the difference between the exposed surfaces and intermediate I and unfolded state D; SAHA, suberoylanilide hydroxamic acid 3578 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS S Kern et al control [5,6] To date, several HDAC inhibitors show potency as antitumor agents, with several drug candidates currently in phase I–III clinical trials [7] Eukaryotic histone deacetylases, as well as their bacterial homologs, have been grouped into four classes, primarily based on sequence similarity [8] Whereas class enzymes (also termed ‘sirtuins’) are NADdependent, class 1, and HDACs are zinc-dependent hydrolases [9] To date, crystal structures of two class enzymes and one class enzyme, as well as of enzyme–inhibitor complexes thereof, are known [10–13] However, no information is currently available on the conformational stability of these enzymes Furthermore, despite the identification of a large number of HDAC inhibitors, the effect of inhibitor binding on enzyme structure and conformational stability of the enzyme has not been analyzed in detail In general, the stability of proteins is an issue of utmost interest in biochemistry and biophysics as well as in industrial enzyme applications The conformational stability of most proteins is surprisingly low, generally between 20 and 60 kJỈmol)1 [14,15] This small overall stability is the result of large contributions from several important converse forces The major destabilizing force is conformational entropy The major stabilizing forces are hydrogen bonding and the hydrophobic effect, which is also responsible for the large change in heat capacity between the unfolded and folded conformations [16,17] For technical applications of enzymes, in most cases maximal stability is desired without losses in activity There are mainly two approaches to stabilize proteins (a) changes in amino acid sequence or (b) specific binding of ions or compounds to the folded conformation The largest increase in conformational stability resulting from a single change in amino acid sequence is the Asn57>Ile mutant of yeast iso-1-cytochrome c by 17.64 kJỈmol)1 [18] Some studies report the stabilizing effects of inorganic ions that specifically bind to the folded conformation of a protein [19–22] For example, Brandts et al used differential scanning calorimetry to measure protein stabilization by ferric ions [23] and highly charged ligands They showed that binding of cytidine 2¢-monophosphate, other nucleotide monophosphates, pyrophosphate and phosphate shifted the transition temperature for ribonuclease thermal unfolding [24] In this study, Brandts et al suggested to use this approach for screening drug candidates for the estimation of binding constants or screening solution conditions to optimize liquid protein formulations with respect to stability Recently, small molecules were found to rescue mutant proteins from degradation and to facilitate trafficking to their site of Inhibitor-mediated stabilization of HDAH action [25–27] These compounds are called chemical chaperones and those compounds which act selectively on a certain pharmaceutical target protein are called pharmacological chaperones Although the precise mechanism of action is not yet completely understood, it is generally assumed that chemical chaperones stabilize a protein conformation capable of escaping the quality control system of the cell [25–27] However, in most of these studies the stabilization of the protein conformation was not measured directly and quantified in terms of free energy Here, we studied the conformational stability of the HDAC class homolog FB188 HDAH, a bacterial HDAC-like amidohydrolase from Bordetella ⁄ Alcaligenes species FB188 [28] FB188 HDAH has been shown to be an excellent model system for HDACs, concerning both structure [13] and function [29,30] The main focus of this report was to investigate the impact of HDAC inhibitors as potential chemical chaperones (i.e stabilizers) as well as zinc and potassium ions on the conformational stability of HDAH Two main denaturation phases of HDAH were differentiated The denaturation occurring at submolar concentrations of the denaturant guanidine hydrochloride (Gdn-HCl) was not affected by inhibitor binding, whereas the denaturation at higher concentrations of Gdn-HCl was strongly affected The existence of at least one conformational intermediate was confirmed by the fact that denaturation of HDAH occurs at a slightly higher denaturant concentration than the loss of enzyme activity Moreover, the investigation of the denaturation and refolding kinetics supports the view that the interconversion between the native and the completely denatured state of HDAH follows a considerably complex mechanism We have shown that the overall conformational stability of HDAH is significantly increased upon binding of the inhibitors cyclopentylpropionyl hydroxamate (CypX) and suberoylanilide hydroxamic acid (SAHA) Data of refolding kinetics demonstrate the strong stabilizing impact of zinc ions, and, to a lesser extent of potassium ions, on the conformational structure of HDAH Results and Discussion Stabilization of conformational structure of HDAH by inhibitors Taking FB188 HDAH as a model of HDACs, we were interested to see whether small-molecule inhibitors would also act as molecular chaperones To elucidate the molecular mechanism of stabilization of protein structure by inhibitor binding, we performed titrations FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3579 Inhibitor-mediated stabilization of HDAH S Kern et al 100 100 - Inhibitor CypX SAHA Enzyme Activity 80 80 60 60 40 40 20 20 Enzyme Activity / % Norm Fluorescence Intensity using Gdn-HCl as the denaturant and analyzed stopped-flow kinetics of the denaturation reaction as well as refolding of HDAH in the absence and the presence of small organic molecule inhibitors Denaturation experiments were performed in the presence of 0–4.5 m Gdn-HCl HDAH showed a biphasic denaturation curve upon increasing the concentration of the denaturant (Fig 1) The protein fluorescence excited at 295 nm and measured at 350 nm originates from five tryptophans of HDAH One of the tryptophans (Trp13) is solvent accessible Two tryptophans (Trp179, Trp191) are buried in the hydrophobic center of the protein, and two further tryptophans (Trp7, Trp192) have limited solvent accessibility The tryptophans with no or limited solvent accessibility presumably possess relatively high quantum yields Upon unfolding, these tryptophans become more exposed to water, which leads to a decrease of their quantum yield and a shift of the maximum of the emission spectrum from 353 nm in the native state to 360 nm in the denatured state The emission maximum at 353 nm did not change at concentrations of Gdn-HCl < 1.5 m In contrast, an unusual decrease in HDAH protein fluorescence intensity was observed at submolar concentrations of Gdn-HCl This decrease in fluorescence was not caused by an artificial contribution of the GdnHCl solution used in all denaturation experiments, as confirmed in a control experiment where the dissolved amino acid tryptophan was titrated with Gdn-HCl (data not shown) Therefore, we conclude that partial 0 c(Gdn-HCl) / M Fig Denaturation curves of 250 nM histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in the absence of inhibitor (squares) and in the presence of 100 lM cyclopentylpropionyl hydroxamate (CypX) (triangles) and suberoylanilide hydroxamic acid (SAHA) (circles) The normalized fluorescence intensity (excitation 295 nm, emission 350 nm) is plotted versus the concentration of the denaturant guanidine hydrochloride (GdnHCl) The unbroken lines (except that for enzymatic activity) are the result of fitting the denaturation data to Eqn (1) The crosses denote the corresponding relative enzyme activity of HDAH in the absence of inhibitor 3580 unfolding of HDAH takes place at submolar concentrations of Gdn-HCl This denaturation phase at low denaturant concentration contributes about 40% to the overall process This study concentrates mainly on the denaturation effect at higher Gdn-HCl concentrations, which, in contrast to the unfolding at submolar concentrations of Gdn-HCl, is clearly affected by inhibitor binding The complete denaturation curve can be fitted using a model function consisting of two addends As the denaturation curve at submolar denaturant concentration cannot be explained by a simple two-state model where the native state denaturates into an intermediate, the first addend just describes the shape of the denaturation phase at submolar denaturant concentration and does not yield parameters with thermodynamic meaning However, the second addend describing the major part of the denaturation curve which is affected by binding of inhibitors directly yields the free energy of unfolding, DGu, of the intermediate in the absence of denaturant and the equilibrium meq value meq is a parameter which reflects the change in compactness of HDAH upon denaturation The parameter is proportional to the surface area buried in the intermediate state I Therefore, the DGu of 17.9 kJỈmol)1 for the major denaturation phase at higher Gdn-HCl concentrations is a lower estimate of the overall conformational stability of free HDAH, being consistent with the conformational stability of most other proteins, which is between 20 and 60 kJỈmol)1 [14,15] There are only rare reports about conformational changes of protein structures at submolar denaturation concentrations The structural changes of horseradish peroxidase and spectrin at submolar concentrations of denaturant are examples reported by Ray et al [31] and Chakrabarti et al [32], although the observed changes in the biophysical parameters were much smaller as compared with the denaturation of HDAH Saturating concentrations of SAHA or CypX were used in all experiments where inhibitors were present The binding constants of SAHA and CypX to HDAH were determined by Riester et al [33], using a competitive binding assay based on fluorescence energy transfer, and are summarized in Table As only the denaturation phase at higher denaturant concentration is affected by the binding of inhibitors, the difference between the free energy of protein unfolding of free and complexed HDAH (DDGu) is identical to the increase of the conformational stability of HDAH by 9.4 and 5.4 kJỈ mol)1 upon binding of SAHA or CypX, respectively (Table 1) This large contribution to the conformational stability is at least one-third of the lower estimate of the conformational stability of the whole protein The FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS S Kern et al Inhibitor-mediated stabilization of HDAH A 1.0 Normalized Fluorescence Table Equilibrium parameters of histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in the absence and the presence of inhibitors K2 denotes the binding constant of the respective inhibitor to HDAH [33] The free energy of unfolding (DGu) from the intermediate to the denatured and the parameter meq were obtained from fitting the data of the equilibrium denaturation curve to Eqn (1) The increase in conformational stability of HDAH, DDGu, upon inhibitor binding is the difference between the free energy of unfolding of HDAH in the absence of inhibitor and in the presence of 100 lM of the noted inhibitor CypX or SAHA 0.8 0.6 0.4 0.2 0.0 DGu (kJỈmol)1) DDGu (kJỈmol)1) No inhibitor + SAHA + CypX – 1.0 0.7 ) 10.3 ± 1.6 ) 11.7 ± 1.5 ) 10.4 ± 1.5 17.9 ± 3.0 27.3 ± 3.5 23.3 ± 3.6 – 9.4 5.4 binding of the hydroxamic acid derivatives to the zinc ion, His142, His143 and Tyr312 within the active site, as well as hydrophobic interactions of the aliphatic chains with Phe152 and Phe208, are believed to contribute to the stabilization of the the major part of the conformational structure of HDAH [13] The meq value of free and complexed HDAH is about )11 kJỈ mol)1Ỉm)1 This is consistent with the assumption that the intermediate, I, is more compact than the denatured protein, D The larger the meq value, the greater the difference between I and D in exposed surface area The magnitude of the meq value is comparable to the overall meq values of other proteins, which range between )2.5 and )18 kJỈmol)1Ỉm)1 [34] The part of the protein that unfolds at submolar denaturant concentrations is quite labile and is not affected upon inhibitor binding To obtain more insight into the mechanism of stabilization by HDAH inhibitors, the refolding and denaturation kinetics in the presence and absence of CypX were investigated If not noted otherwise, 10 mm K+ was used in these experiments The refolding kinetics in the presence of 0.5 mm Zn2+ and 100 lm CypX was slightly slower when compared with the kinetics in the absence of CypX At 50 lm Zn2+, the overall refolding kinetics was markedly slower, showing a sigmoidal increase Upon the addition of 100 lm CypX, again the refolding kinetics was only slightly slower than in the absence of CypX This negative impact of CypX on refolding can be explained by the Zn2+ dependency of HDAH refolding As pointed out in the following section, the refolding kinetics is strongly dependent on the concentration of zinc ions (Fig 2A,B) CypX is a hydroxamate derivative and hydroxamates are known to complex divalent cations, such like Zn2+ Thus, free CypX is able to bind Zn2+ ions, which otherwise would accelerate the refolding of B Normalized Fluorescence meq (kJỈmol)1ỈM)1) 1.0 0.8 0.6 0.4 0.2 0.0 C Normalized Fluorescence Inhibitor K2 (106ỈM)1) 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 Time / s 40 50 60 Fig Refolding kinetics of histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) (A) in the presence of 10 mM KCl and different concentrations of Zn2+ (0 mM, blue; 0.05 mM, red; 0.5 mM, dark green; 1.0 mM, black) and (B) in the presence of different concentrations of Zn2+ and K+ ions (0 mM Zn2+ + mM K+, brown; mM Zn2+ + 10 mM K+, blue; mM Zn2+ + mM K+, orange; mM Zn2+ + 10 mM K+, black) and (C) in the presence of 10 mM K+ and in the presence of different concentrations of Zn2+ ions in the absence or the presence of 100 lM inhibitor cyclopentylpropionyl hydroxamate (CypX) (0.05 mM Zn2+ ) CypX, red; 0.05 mM Zn2+ + Cyp X, magenta; 0.5 mM Zn2+ ) CypX, dark green; 0.5 mM Zn2+ + CypX, light green) The normalized fluorescence of stopped-flow experiments is plotted versus time First, the enzyme was denatured using M guanidine hydrochloride (Gdn-HCl) Then, refolding was initiated by diluting the denatured enzyme in Tris buffer, pH 8.0, to a final Gdn-HCl concentration of 0.6 M HDAH Under these conditions of refolding, the competition between HDAH and CypX for Zn2+ binding causes a slightly retarded refolding kinetics FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3581 Inhibitor-mediated stabilization of HDAH S Kern et al In contrast, CypX has a significant impact on the denaturation kinetics of HDAH in 15 mm Tris ⁄ HCl buffer, pH 8.0 (Fig 3) At 2.8 m Gdn-HCl the amplitudes of the denaturation curves were almost the same in the absence and the presence of 100 lm CypX, but the denaturation kinetics in the presence of CypX was significantly slower (Fig 3A) At 3.2 m, the denaturation curves could not be distinguished (Fig 3B) This would be expected if the binding of CypX to HDAH is inhibited in the presence of 3.2 m Gdn-HCl This would also explain why the effect of CypX on the unfolding kinetics in the presence of 2.8 m Gnd-HCl is weak compared with the strong effect of CypX on the stability of the protein Based on these results, we conclude that the inhibitor CypX can stabilize the conformational structure of HDAH by decelerating the denaturation process Hydroxamate-derived inhibitors Normalized Fluorescence A 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 10 10 Time / s Normalized Fluorescence B 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 Time / s Fig Denaturation kinetics of 500 nM histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in Trisphosphate buffer, pH 8.0, in the presence of 2.8 M (A) or 3.2 M (B) guanidine hydrochloride (Gdn-HCl) The normalized fluorescence of the intrinsic tryptophans of HDAH is plotted versus time At the lower concentration of Gdn-HCl (A) the difference between the denaturation kinetics in the presence of 100 lM cyclopentylpropionyl hydroxamate (CypX) (red) and in the absence of inhibitor (blue) becomes visible 3582 can even be contraproductive in refolding experiments of Zn2+-dependent enzymes such as HDAH because hydroxamate complexes Zn2+ ions, which accelerate refolding dramatically (see the next section) Impact of Zn2+ and K+ ions on the refolding of HDAH The crystal structure of HDAH contains one Zn2+ ion at the bottom of the active site and two K+ ions in the neighbourhood of the active site [13] The importance of Zn2+ and K+ ions for the conformational structure of HDAH was investigated by measuring the refolding kinetics of HDAH (Fig 2A,B) in the presence or absence of these cations The kinetics of HDAH refolding in the presence of 10 mm K+ is strongly dependent on Zn2+, which underlines the pivotal role of Zn2+ within the active site of HDAH for the conformational stability of HDAH (Fig 2A and 4A) If Zn2+ is present at 0.5 or mm, the folding of the polypeptide chain into the correct orientation of the enzyme is facilitated as a result of the interactions between the zinc ion and the adjacent amino acids Asp180, Asp268 and H182 The kinetics in the presence of 0.5 mm Zn2+, 10 mm K+ and a final concentration of 0.6 m Gdn-HCl at 21°C behaves like a single exponential with a time constant of 2.3 s With decreasing concentrations of Zn2+, the refolding kinetics becomes strongly retarded At lower Zn2+ concentrations the kinetics changes into a sigmoidal behaviour, indicating a more complex mechanism of the refolding process with at least one additional intermediate in the absence or at lower concentrations of Zn2+, which becomes rate limiting Perhaps the mechanism is followed also in the presence of 0.5 mm Zn2+, where the time course of the refolding kinetics can be described by only one exponential In this case it could be assumed that the first process will be accelerated by Zn2+, such that this step is no longer rate limiting Another explanation would be that the refolding mechanism would change in the presence of Zn2+ Refolding rates at mm Zn2+, on the other hand, gave rise to clear double-exponential decays with two well-separated phases Both effects – the slow effect and the fast effect – strongly depend on the final Gdn-HCl concentration (Fig 4B) Such additional effects might be ascribed to one of three phenomena: (a) transient aggregation during folding [35,36] (b) cistrans isomerization (e.g cis-trans isomerization about prolyl-peptidyl bonds) [37–39] or (c) the formation or decay of folding intermediates [40] Aggregation can be ruled out, as the refolding rate constant did not vary significantly with protein concentration over a 10-fold FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS S Kern et al A Inhibitor-mediated stabilization of HDAH 0.0 ln kobs -1.0 -2.0 -3.0 -4.0 0.0 B 0.5 1.0 1.5 c(Gdn-HCl) / M 2.0 2.5 1.0 2.0 2.5 3.0 2.0 ln kobs 1.0 0.0 -1.0 -2.0 -3.0 -4.0 0.0 0.5 1.5 c(Gdn-HCl) / M Fig Refolding rate constants of histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) The logarithm of the refolding rate constants is plotted versus the concentration of guanidine hydrochloride (Gdn-HCl) The kinetics were measured in the presence of 10 mM KCl and the denoted concentrations of ZnCl2 The data were fitted to a two-state model (dashed line, Eqn 5) and to the on-pathway intermediate model (solid line, Eqn 7) The kinetic parameters are summarized in Table (A) The concentrations of ZnCl2 are mM (open squares), 0.05 mM (filled squares), 0.5 mM (circles) and 1.0 mM (diamonds) Only the rate constant of the main slow increasing effect is shown in panel A (B) At mM Zn2+ an additional fast effect with increasing fluorescence intensity becomes visible Both the rate constants of the fast (triangles) and the slow (diamonds) processes are plotted versus the concentration of Gdn-HCl In the following, we will concentrate on the slow phase and analyse the data according to the three-state approach outlined in the experimental procedures Additional information about the accessible surface of the conformational structures during the refolding process can be obtained from the dependence of the refolding rate constants on the final concentration of Gdn-HCl [42,43] Figure 4A shows plots of ln(k) of the slow refolding process versus Gdn-HCl concentration at different concentrations of Zn2 + and 10 mm K + In the absence and the presence of 50 lm Zn2 + at Gdn-HCl concentrations below 0.6 m, the rate constants were about one order of magnitude smaller compared with the refolding kinetics in the presence of 0.5 and 1.0 mm Zn2 + (Fig 4A) At final Gdn-HCl concentrations higher than 0.6 m, no temporal change in fluorescence intensity was observed in the presence of or 50 lm Zn2 + This means that no measurable refolding occurs at these concentrations of Zn2 + and GdnHCl As seen inFig 4A, the logarithmic refolding rate constants in the presence of 0.5 and mm Zn2 + display a linear dependence at Gdn-HCl concentrations higher than 1.0 m This can be taken as a two-state transition from the denatured to an intermediate state, which dominates the folding reaction at concentrations of > m Gdn-HCl The slope, which is proportional to the difference between the accessible protein surface after and before refolding, is negative, which is consistent with the assumption that the intermediate is more compact than the denatured protein At concentrations below m Gdn-HCl, the plot of ln(kobs) versus denaturant concentration shows a clear rollover effect in which the slope of the curve decreases significantly (Fig 4) This behaviour of the folding kinetics suggests that an intermediate accumulates transiently during refolding [44] All curves of logarithmic rate constants versus denaturant concentration could satisfactorily be fitted to both the on-pathway or the off-pathway models (Eqns and 9; Fig 4) On the basis of the kinetic data, it cannot be distinguished between productive on-pathway intermediate I: KI kf D , I , N; ku range (data not shown) The fast phase, at mm Zn2+, is also much faster than a conventional isomerization step, which usually has reaction rates in the order of 10)2 to 10)4Ỉs)1 [41] And, in contrast to our observation, cis-trans isomerization processes not depend on the final denaturant concentration of refolding These arguments point again to at least one, probably more, additional intermediates and off-pathway intermediate C: KC kf C , D , N: ku The fitted parameters of both models are summarized in Table Further experiments are required to identify whether there is an on-pathway or an off-pathway intermediate Taking into account the sigmoidal shape FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3583 Inhibitor-mediated stabilization of HDAH S Kern et al Table Kinetic parameters of histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) refolding in the presence of noted concentrations of Zn2+, c(Zn2+) Kinetic parameters of HDAH refolding are shown in the presence of 0.5 and 1.0 mM Zn2+ The parameters were obtained by fitting the data of ln(kobs) versus the concentration of Gdn-HCl, c(Gdn-HCl), to different folding models (see Eqns 5, and 9) KC, ratio of off-pathway intermediate C and denatured state D; kf, the folding rate in the absence of denaturant; KI, ratio of on-pathway intermediate I and denatured state D; mf reflects the change in solvent-accessible area in the process of refolding; mi, reflects the change in solvent-accessible area in the transition from the denatured to the intermediate state c(Zn2+) Folding model kf (s)1) 0.5 mM Two-state Three-state, Three-state, Two-state Three-state, Three-state, Two-state Three-state, Three-state, 9.3 0.38 58 5.2 0.39 10.1 34.5 2.1 41 mM Slow phase mM Fast phase on-path off-path on-path off-path on-path off-path ± ± ± ± ± ± ± ± ± mf (kJỈmol)1ỈM)1) 4.6 0.08 51 1.6 0,16 6.0 8.6 4.9 22 KI KC mI (kJỈmol)1ỈM)1) )8.6 0.9 11.7 ) 5.3 3.9 )6.2 )5.9 13 )6.3 – 152 ± 113 – – 26 ± 12 – – 153 ± 112 – – 26 ± 12 – – 19 ± 37 – )12.5 )12.5 – )10.0 )10.1 – )20 )20 of refolding timecourses at very low Zn2+ concentrations (Fig 2), the additional fast phase at mm Zn2+ (Fig 4B) and the additional equilibrium denaturation phase at low-denaturant concentration (Fig 1), which cannot be explained by a simple transition from the native to one transition state, it is evident that folding of HDAH is rather complex and must pass through more than just one intermediate Without Zn2+ and K+, no increase in fluorescence can be detected within 60 s (Fig 2B) In the presence of 10 mm K+, but in the absence of Zn2+, a slow sigmoidal increase in fluorescence intensity can be measured (Fig 2B) In summary, the refolding mechanism appears to be strongly dependent on Zn2+ and to a lesser extent on K+, which underlines the importance of both cations, not only for the function of the enzyme but also for the correct conformational structure There is strong evidence that HDAH folding involves more than one intermediate It was shown that the known inhibitors SAHA and CypX are capable of stabilizing the conformational structure of HDAH Judging from free energies of unfolding, the conformational stability of a complex between these inhibitors and HDAH is more than 30% higher than the stability of unbound HDAH We suspect that hydroxamate-type HDAC-inibitors such as CypX or SAHA not only hinder substrates to obtain access to the active site, but rather may even freeze HDACs in catalytically unproductive conformations In this connection it is interesting to note that the association kinetics of N-(2-furyl)acryloyl-hydroxamic acid and HDAH can only be satisfactorily described by a biphasic exponential model [31], suggesting a multistep binding process, including conformational changes of the enzyme If we now assume a similar behavior of eukaryotic HDACs upon inhibitor binding, 3584 ± ± ± ± ± ± ± ± ± 1.0 1.3 1.4 0.5 2,6 0.8 0.5 17 1.0 – 19 ± 37 – ± 0.7 ± 0.7 ± 2.1 ± 2.1 ± 17 ± 17 it is tempting to speculate that inhibitor-induced conformational changes of HDACs are responsible for breaking up corepressor complexes as, for example, described in the case of acute myelocytic leucemia [45] Furthermore, our results support the assumption that specific ligands of proteins within cells may act as molecular chaperones by stabilizing a protein conformation capable of escaping the quality control system of the cell A better understanding of the impact of inhibitor binding on the stability of target proteins (e.g HDAH) may result in new concepts for lead structures Experimental procedures Materials His-tagged FB188 HDAH was prepared as described previously [28] SAHA, CYPX and phenylpropionyl hydroxamate were synthesized according to standard methods [13,46,47] If not stated otherwise, the denaturation experiments were carried out in Tris-phosphate buffer consisting of 250 mm sodium chloride, 250 lm EDTA, 15 mm TrisHCl and 50 mm potassium hydrogen phosphate, pH 8.0 Enzyme activity assay FB188 HDAH exhibits amidohydrolase [28] and esterase activity [48] Amidohydrolase activity was assayed in the two-step assay [49,50] As trypsin activity is required in this type of assay, and trypsin rapidly denatures upon addition of denaturant, the two-step assay was not suited for activity measurements in samples containing Gdn-HCl Esterase activity was monitored using 4-methylcoumarin-7-acetate as a substrate [48] This type of assay was used for samples containing Gdn-HCl FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS S Kern et al Denaturation experiments using guanidine hydrochloride In the denaturation experiments, 250 nm HDAH in phosphate buffer, pH 8.0, were titrated with increasing amounts of a solution of m Gdn-HCl in the same buffer All experiments were carried out at 21 ± 0.2 °C The denaturation of the protein was followed by measuring its tryptophan fluorescence The tryptophans of HDAH were excited at 295 nm and their fluorescence emission was measured at 350 nm in a Hitachi (Tokyo, Japan) F-4000 spectrofluorometer using and 10 nm slits, respectively After each addition of Gdn-HCl and thorough mixing, the fluorescence was measured until the signal was constant within ± 1% for at least 30 s This value was considered to represent the unfolding equilibrium and was plotted against the corresponding Gdn-HCl concentration The resulting graph is called the denaturation curve Data analysis of equilibrium protein denaturation curves The fitting of equilibrium unfolding curves is described in detail by Santoro & Bolen [51] and directly gives thermodynamic parameters of the corresponding denaturation curves The fitting function was slightly complemented to account for the additional denaturation phase at submolar denaturant concentrations The fluorescence signal as a function of denaturant concentration was fitted to the following expression: Fẳ A ỵ exphb ẵGdn HCl IC50 ịi FI ỵ FD exphDGu ỵ meq ẵGdn HClị=RTi ; 1ị ỵ ỵ exphDGu ỵ meq ẵGdn HClị=RTi where DGu is the free energy of unfolding of an intermediate in the absence of denaturant; meq is the equilibrium m-value, which is proportional to the difference between the exposed surfaces of intermediate I and unfolded state D; FI and FD are the fluorescence signals of I and D; A, b and IC50 are the amplitude, the steepness and the inflection point, respectively, and used as arbitrary parameters to describe the contribution of the additional denaturation process at submolar denaturant concentration; [Gdn-HCl] is the concentration of Gdn-HCl and RT is the product of the gas constant and temperature All equilibrium and kinetic data were fitted using the program scientist from micromath (St Louis, MO, USA) Stopped-flow kinetics All measurements of displacement and renaturation kinetics were carried out on a Bio-Logic (Claix, France) MOS-250 Inhibitor-mediated stabilization of HDAH Stopped-Flow instrument equipped with a 150 W xenon mercury light source attached to a manual monochromator on an optical bench The connection to the Bio-Logic Stopped-Flow instrument was performed through a fiber optic specially designed to match the stopped-flow cuvette dimensions The signal detection was performed by a photomultiplier directly mounted on the stopped-flow and connected to its control unit The photomultiplier was attached at 90° of the light source allowing for fluorescence measurements The HDAH tryptophans were excited at 295 nm A polystyrene filter was installed in front of the photomultiplier tube to reject scattered light The photomultiplier control unit was connected to a 16-bit A ⁄ D board installed in a PC driven by the acquisition and analysis software bio-kine32 (Claix, France) The core unit of the instrument is a temperature-controlled metal block containing three syringes and a mixing chamber The syringes are driven by precise and robust high-speed stepping-motors The dead time of the apparatus was calculated to be below ms The temperature was controlled at 21 ± 0.2°C Stopped-flow data were fitted to either a monophasic  ! t ỵB 2ị Ftị ẳ A1 À exp À s1 or a biphasic  !  ! t t ỵ A2 exp ỵ B 3ị Ftị ẳ A1 exp s1 s2 exponential model by using a nonlinear least-square fitting procedure integrated in the analysis software biokine32 F(t) is the observed fluorescence of the protein at time t after the start of the reaction and B is the background signal A1 and A2 are the amplitudes of two exponential changes, and s1 and s2 are their respective kinetic time constants The refolding kinetics were initiated by mixing buffer consisting of 15 mm Tris ⁄ HCl, pH 8.0, and denoted concentrations of Zn2+, K+ ions or CypX, and completely denatured HDAH dissolved in the same buffer in the presence of m Gdn-HCl The denaturation kinetics were carried out by mixing 500 nm HDAH (final concentration) in 15 mm Tris ⁄ HCl, pH 8.0, in the absence or presence of 100 lm CypX and 15 mm Tris ⁄ HCl buffer, pH 8.0, containing denoted concentrations of Gdn-HCl Data analysis of refolding kinetics The analysis of the kinetic data is based on a linear relationship between the log of microscopic rate constants and the denaturant concentration The following equations were adapted from Mogensen et al [52] and slightly modified to fit rate constants determined from stopped-flow experiments Under the condition of the experiments the contribution of unfolding could be disregarded FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3585 Inhibitor-mediated stabilization of HDAH S Kern et al Two-state folding: kf D,N ð4Þ À À À Á ln kobs ẳ ln exp ln kf ỵ mf ẵGdn HCl =RT 5ị ku where in this simple two-state case D denotes the denatured state and N denotes the native state of the protein; kobs is the observed folding rate; kf is the folding rate in the absence of denaturant and mf is the corresponding m-value which reflects the change in solvent-accessible area in the process of refolding For a simple two-state folding mechanism a plot of ln kobs versus the concentration of GdnHCl, c(Gdn-HCl), is expected to be linear over the whole range of denaturant concentration The accumulation of on- or off-pathway intermediates during folding will give rise to deviations from linearity, particularly at low denaturant concentrations The models for folding mechanisms over in- or off-pathway intermediates follow (A) Folding over an on-pathway intermediate: KI kf D,I ,N ku ln kobs ẳ ln 6ị ! exp ln kf ỵ mf ẵGdn HCl=RT ; 7ị ỵ exp ln KI ị ỵ mI ẵGdn HCl=RT ịị where I is an on-pathway 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mutation Biochemistry 43, 3357–3367 Supplementary material The following supplementary material is available online: Fig S1 Fitting results of refolding kinetics at different concentrations of zinc ions This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS ... integrated in the analysis software biokine32 F(t) is the observed fluorescence of the protein at time t after the start of the reaction and B is the background signal A1 and A2 are the amplitudes of. .. Ray et al [31] and Chakrabarti et al [32], although the observed changes in the biophysical parameters were much smaller as compared with the denaturation of HDAH Saturating concentrations of. .. the active site, as well as hydrophobic interactions of the aliphatic chains with Phe152 and Phe208, are believed to contribute to the stabilization of the the major part of the conformational