Crystal Structure of a Histone Deacetylase Homologue from

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Crystal structure of a histone deacetylase homolog from Pseudomonas aeruginosa Andreas Kraemer, Thomas Wagner, Özkan Yildiz, and Franz-Josef Meyer-Almes Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00613 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016

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Biochemistry

1

Crystal structure of a histone deacetylase homolog

2

from Pseudomonas aeruginosa

3

Andreas Krämerǂ, Thomas Wagnerǂ, Özkan Yildiz£, Franz-Josef Meyer-Almesǂ*

4 5

ǂ

University of Applied Science, Department of Chemical Engineering and Biotechnology, 64295

6 7 8 9

Darmstadt, Germany £

Max Planck Institute of Biophysics, Department of Structural Biology, 60438 Frankfurt am Main, Germany

10 11 12 13 14 15 16

ACS Paragon Plus Environment

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Biochemistry

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17

KEYWORDS

18

Histone deacetylase, HDAC, Pseudomonas aeruginosa, Acetate, Amidohydrolases, X-Ray

19

Crystallography, Histone Deacetylase Inhibitors, Lysine Deacetylation, Histone deacetylase like

20

amidohydrolase,

21

amdidohydrolase, APAH

HDAH,

Histone

deacetylase

like

protein,

HDLP,

acetylpolyamine-

22 23

ABBREVIATIONS

24

HDAC, histone deacetylase; HDAH, histone deacetylase-like amidohydrolase; HDLP, histone

25

deacetylase-like protein; KDAC, lysine deacetylase; APAH, acetylpolyamine amidohydrolase;

26

SAHA,

27

nonanamide; TFA, trifluoroacetate; Ac, acetate; AMC, 7-Amino-4-methylcoumarin; boc, tert-

28

butyloxycarbonyl; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, 2-

29

Amino-2-hydroxymethyl-propane-1,3-diol; RMSD., root mean square deviation; PFSAHA,

30

2,2,3,3,4,4,5,5,6,6,7,7‐dodecafluoro‐N‐hydroxy‐N'‐phenyloctanediamide

N-Hydroxy-N′-phenyloctandiamid;

SATFMK,

9,9,9-trifluoro-8-oxo-N-phenyl-

31 32 33 34 35 36 37 38


2

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Biochemistry

39 40

ABSTRACT

41

Despite the recently growing interest in the acetylation of lysine residues by prokaryotic

42

enzymes, the underlying biological function is still not well understood. Deacetylation is

43

accomplished by proteins which belong to the Histone deacetylase (HDAC) superfamily. In this

44

report we present the first crystal structure of PA3774, a histone deacetylase homolog from the

45

human pathogen Pseudomonas aeruginosa with high homology to class IIb HDACs. We solved

46

the crystal structure of the ligand-free enzyme and protein-ligand complexes with a

47

trifluoromethylketone inhibitor and the reaction product acetate. Moreover, we produced loss of

48

function mutants and determined the structure of the inhibitor free PA3774H143A mutant, the

49

inhibitor-free PA3774Y313F mutant and the PA3774Y313F mutant in complex with the highly

50

selective hydroxamate inhibitor PFSAHA. The overall structure reveals that the exceptionally

51

long L1-loop mediates the formation of a tetramer composed of two “head-to-head” dimers. The

52

distinctive dimer interface significantly confines the entrance area of the active site suggesting a

53

crucial role for substrate recognition and selectivity.

54 55 56 57 58 59 60


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INTRODUCTION

62

Histone deacetylases (HDAC), acetylpolyamine-amidohydrolases (APAH) and acetoin

63

utilization Proteins (AcuC) belong to an ancient protein superfamily known as the histone

64

deacetylase superfamily 1. In the past years especially histone deacetylases raised much attention

65

due to their important roles in the cell cycle and differentiation. For instance, deregulation of

66

these enzymes can contribute to the development of cancer

67

novel therapeutic target for chemo therapy which led to the approval of new drugs for the

68

treatment of cutaneous T-cell lymphoma such as Vorinostat and Romidepsin 4, 5. The HDACs are

69

classified in four groups based on their sequence and domain organization. Class Ι, IIa, IIb and

70

IV are known as zinc dependent “classical” histone deacetylases, whereas class III HDACs or

71

sirtuins constitute an atypical NAD+ dependent class 6. According to sequence analysis class Ι,

72

IIa, IIb, IV share very similar catalytic domain organization and differ mainly in the length of the

73

N- and C-termini. Class Ι HDACs consist of 370-500 residues, class II enzymes have 660-1220

74

residues and class IV which only consist of HDAC11 that shows characteristics of both class Ι

75

and class II 7. Interestingly, HDACs have also been shown to be involved in the deacetylation of

76

acetylated lysine residues in non-histone proteins 8. For this reason they are sometimes called

77

lysine deacetylases (KDACs) with respect to their basic function 9. It has been further suggested

78

that APAHs and AcuCs are the precursor enzymes of HDACs since prokaryotic cells have no

79

histones. Yet, few prokaryotic enzymes with high homology to zinc dependent HDACs have

80

been studied before. Histone deacetylase-like protein (HDLP) from Aquifex aeolicus

81

the highest homology to class Ι HDACs, whereas histone deacetylase-like amidohydrolase

82

(HDAH) from Bordetella/Alcaligenes was assigned to HDAC class IIb

83

prokaryotic studies have shown that APAHs catalyze the deacetylation of acetylated polyamines

2, 3

. In this context, they became a

11

10

exhibits

. In addition,


4

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Biochemistry

84

12, 13

, whereas the reactions catalyzed by AcuC proteins are not known 1. Moreover, to this date

85

there are several structures available for human members of the HDAC family including

86

members of class I: HDAC1

87

and HDAC7 21 and recently class IIb: HDAC6 22, 23.

14

, HDAC2

15, 16

, HDAC3

17

, HDAC8

18, 19

; class IIa: HDAC4

20

88

P. aeruginosa is a gram-negative bacterium with one of the largest sequenced bacterial

89

genome until now consisting of 6.3 million base pairs. It shows a vast variety of metabolic

90

pathways and is able to adapt to a wide range of different environments

91

aeruginosa has multiple defense strategies against many structurally diverse antibiotics and is

92

indeed one of the most relevant human pathogens associated with a wide spectrum of infections,

93

especially in immunosuppressed patients. It is particularly associated with cystic fibrosis,

94

infection of the urinary-tract and bacteremia in burn victims 25.

24

. Moreover, P.

95

In the genome of P. aeruginosa three enzymes with sequence similarity to HDACs were

96

identified: PA0321, PA1409 and PA3774 as referenced in the Pseudomonas genome database 26.

97

Although all three were predicted to be acetylpolyamine-amidohydrolases (APAHs)

98

work demonstrated that only PA0321 and PA1409 are capable of deacetylating acetylated

99

polyamines, whereas PA3774 shows no significant activity 27. On the contrary, PA3774 exhibits turnover

rates

artificial

HDAC

substrates

containing

acetylated

recent

100

high

101

trifluoracetylated lysine residues, thus proposing a role as a protein deacetylase with an hitherto

102

unknown substrate

103

strain PA01. We present a total of six crystal structures, including the ligand-free enzyme, the

104

enzyme in complex with acetate, a complex with a trifluoromethylketone inhibitor, and

105

additional three loss-of-function mutant structures: PA3774H143A and PA3774Y313F without

106

ligands and the complex between PA3774Y313F and the hydroxamate inhibitor PFSAHA. The

27

against

26

and

. We here present the crystal structure of PA3774 from the P. aeruginosa


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observed quaternary structure suggests that tetramer assembly is essential for substrate

108

recognition and selectivity. The structural information on the active site extends our

109

understanding of class II HDACs and provides a solid base for the rational design of specific and

110

potent inhibitors for this class. Furthermore, our mutational studies demonstrate the important

111

role of catalytic core residues and invoke the hypothesis of different deacetylation mechanisms

112

in class Ι and class IIb HDACs.

113 114

MATERIAL AND METHODS

115

Reagents. Standard chemicals and solvents for activity test and crystallization were purchased

116

at the highest available purity level from Sigma-Aldrich (USA), Merck (Germany), Roth

117

(Germany). Boc-Lys(Ac)-AMC and Boc-Lys(TFA)-AMC from Bachem (Switzerland),

118

Paratone-N from Hampton.

119

Overexpression, purification and mutagenesis. The enzyme PA3774 was overexpressed and

120

purified as published elsewhere 28 (For detailed chromatograms and SDS PAGEs see supporting

121

online material Fig. S1). The PA3774H143A, PA3774H144A, PA3774Y313H and PA3774Y313F

122

mutants were prepared using the QuickChange site-directed mutagenesis Kit (Stratagene), the

123

pET21a(+)/PA3774-CPD plasmid as template and the oligonucleotide primers listed in the

124

supplementary data (Fig. S2).

125

Following PCR mutagenesis and DpnI digestion, the resulting plasmids were used to transform

126

E. Coli XL1-Blue competent cells. After transformation the cells were plated on LB-agar plates

127

containing 100 µg/mL ampicillin. Plasmid DNA isolated from single colonies of transformants

128

was sequenced to verify the mutations by the sequencing service of LMU Munich. Plasmids

129

containing the desired mutations were then used to transform E. coli BL21(DE3) cells.


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Biochemistry

130

Overexpression and purification of the mutated enzymes was identical as described for the native

131

enzyme.

132

Crystallization and data collection. Crystals of native and mutated PA3774 were grown by

133

hanging drop vapor diffusion against 500 µl reservoir (Fig. S3). The reservoir solution contained

134

0.5 M K2HPO4, 0.5 M Na2HPO4, 0.1 M (NH4)2SO4 at a final pH of 7.5. The reservoir solution

135

and protein (6 mg/ml dissolved in 20 mM HEPES pH 8, 50 mM NaCl, 2 mM TCEP) were mixed

136

in a 1:1 ratio to a final volume of 4 µl. Crystals grew in two to four days at 20°C. The obtained

137

crystals were soaked with the inhibitor added in 10 fold molar excess directly to the drop and

138

incubated for at least 48 h. To obtain the PA3774-acetate complex the crystals were soaked with

139

50 mM ammonium-acetate. The crystals were transferred into Paratone-N and flash-frozen in

140

liquid nitrogen. Diffraction data were collected on beamline PXII at the Swiss Light Source

141

(SLS, Villigen, Switzerland).

142

Structure determination and refinement. Diffraction data were indexed and integrated using 29

and scaled with AIMLESS 30. The structure was solved by molecular replacement

143

iMOSFLM

144

with MOLREP 31 using one monomer of the HDAH structure from Bordetella/Alcaligenes strain

145

FB188 (pdb-id 1ZZ0) as a model. Model building was carried out in COOT 32 and REFMAC5 33

146

was used for refinement. All used programs belong to the CCP4 suite 34. Water molecules were

147

added automatically with COOT and afterwards checked manually for reasonably hydrogen

148

bonding. All Figures were created with PyMOL.

149 150

Enzyme activity assay. Enzyme activity was determined following the standard fluorogenic 35

151

enzyme activity assay in

. Briefly, the cleavage of trifluoroacetate or acetate by the enzyme

152

allows trypsin to release the fluorophore from the substrate, thereby resulting in a shift in


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fluorescence (Ex.: 340 nm Em.: 460 nM). Tests were run at room temperature with 100 nM

154

enzyme and 0.5 mg/mL trypsin solved in assay buffer (20 mM Tris-HCl pH 8, 50 mM NaCl,

155

0.001 % Pluronic). Boc-Lys(Ac)-AMC and Boc-Lys(TFA)-AMC at a concentration of 20 µM

156

were used as fluorogenic substrates. Because trypsin does not digest the enzyme during the test

157

procedure the activity assay can be run in a continuous manner and the linear slope of the

158

fluorescence signal is used to determine the enzyme activity. The experiment was carried out in a

159

PheraStar Fluorescence Spectrometer (BMG Labtech) and data were analyzed with the program

160

Graph Pad Prism.

161 162

RESULTS

163

Overall structure. PA3774 and all variants crystallized in the tetragonal space group P41212

164

with two molecules in the asymmetric unit (Table 1) forming a “head-to-head” homo dimer with

165

a protein-protein interface of about 2090 Å2. The electron density could be interpreted for

166

residues 2-374 of one monomer and 2-379 of the other out of a total number of 384 possible

167

residues. The enzyme core adopts an open α/β fold which is characteristic for this class of

168

enzymes

169

and two smaller antiparallel β-turns (Fig. 1).

18, 36, 37

. It consists of eight central stranded parallel β-sheets surrounded by 14 helices

170

Oligomerization. The unit cell of the tetragonal crystals consists of 8 “head-to-head” dimers

171

described for the asymmetric unit. In here, there are two “head-to-head” dimers in very close

172

distance to each other forming a tetramer. Multi angle laser light scattering (MALLS)

173

experiments following analytical size exclusion chromatography suggest that the tetramer is the

174

relevant quaternary structure in solution (see Fig. S4 and S5). The tetrameric structure is also in

175

line with PISA analysis

38

(for detailed analysis see Fig. S6). However, since the interface area


8

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Biochemistry

176

between the two “head-to-head” dimers is with 2090 Å2 much larger compared to each of the

177

other interfaces between subunits which are about 685 and 407 Å2, the structure is better

178

described as a dimer of dimers. The tetramer has therefore the dihedral point group D2 (also

179

noted as 222). A closer look at the crystal structure reveals that the elongated L1-Loop inside the

180

residues 15-42 plays a pivotal role in the stabilization of the highly symmetric PA3774 tetramer.

181

Every L1-Loop in each subunit points to the symmetry center of the tetramer where all subunits

182

are in contact with each other (Fig. 1D and Fig. S6). A similar assembly can be found in the

183

crystal structures of the related enzyme HDAH from Bordetella/Alcaligenes (pdb-id 1ZZ0) 39, 40

184

(Fig. 2). In this enzyme a similar elongated L1-loop is found. The importance of this long loop

185

region for tetramerization becomes evident by the monomeric structures of human HDAC8 18, 41

186

which has only 4 residues in the corresponding loop (Fig. 2 and 3). Moreover, the oligomeric

187

state and especially the head-to-head dimer interface have a direct influence on the active site. A

188

large area of the entrance cavity in each monomer gets partially blocked by another monomer

189

resulting in a substantially narrowed entrance surface (Fig. 4) suggesting a crucial role for

190

substrate recognition and selectivity.

191

Catalytic center. The catalytic center shows the typical motif observed for many enzymes of

192

the histone deacetylase superfamily. The catalytically zinc ion is trigonal bipyramidal-

193

coordinated by the side chains of D181, H183, D269 and a corresponding ligand. Close to the

194

zinc ion there are three conserved catalytic residues H143, H144 and Y313 which make

195

hydrogen bonds with bound ligands. Single mutations of these residues abolish completely the

196

activity (Table 2). We further localized two octahedrally coordinated potassium ions in PA3774.

197

These monovalent cation sites are also found in other HDACs and HDAC-like proteins

198

potassium ion is coordinated by the main-chain carbonyl of D179, D181, H183, L203 and by the

42

. One


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199

hydroxyl and carboxyl side chain of S203 and D179, respectively. This potassium ion seems to

200

keep the zinc-coordinating residues in position. Especially the hydrogen bridges between the

201

amino acid pairs D179-H143 and H144-N183 are important for the mechanism (Fig. 5A) and

202

found in HDAH, APAH and in most class II HDACs

203

asparagine pair is replaced by histidine-aspartate40. The other potassium ion is coordinated by the

204

main chain carbonyl of V198, Y192, R195, F227 and two water molecules. In contrast to the

205

potassium ions the occupancy for the zinc ion is lower (60-70%), probably due to the absence of

206

zinc ions the crystallization buffer.

207

PA3774-acetate complex. The complex with the reaction product acetate was achieved by

208

soaking the crystals with ammonium acetate in reservoir solution (final concentration 50 mM).

209

The 1.70 Å structure allows a precise interpretation of nearly all side chains and backbone

210

interactions. In this complex the acetate anion coordinates the zinc ion with both oxygen atoms

211

(Fig. 5A), resulting in a trigonal bipyramidal coordination of the zinc ion. The observed binding

212

is almost identical to the acetate bound HDAH structure from Bordetella/Alcaligenes (pdb-id

213

1ZZ0) 40.

214

PA3774–SATFMK complex. The trifluoromethylketone inhibitor SATFMK was soaked in a

215

tenfold molar excess to the protein and the structure was solved at 1.71 Å. The inhibitor has a

216

remarkably high affinity towards the enzyme which is represented by an IC50 value of 9.7 nM 28

217

(Fig. S13). The observed electron density indicates that the ketone inhibitor binds in its gem-diol

218

form in a bidentate fashion (Fig. 5B and Fig. S7). The overall binding geometry of the zinc ion is

219

described best by a trigonal bipyramid. The two oxygen zinc distances are almost identical, with

220

2.08 Å and 2.06 Å respectively. The binding of gem-diol mimics the tetrahedral transition state

221

analogue during the deacetylation. One oxygen of the gem-diol accepts hydrogen bonds from

13, 20, 40

. In class Ι HDACs the histidine-


10

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Biochemistry

222

H144 and H143, whereas the other one from Y313. The high affinity of trifluoromethylketones

223

can be ascribed further to favorable interaction of this group with the buried binding pocket. The

224

fluorine atoms form orthogonal multipolar interactions with the side chain hydroxyl group of

225

Y313, the backbone amide group of G311, the thiol group of C154 as well as van-der-Waals

226

contacts with P141. The binding pocket around the catalytic center is highly conserved in class

227

IIa HDACs and class IIb HDACs. A similar binding was observed for the same inhibitor bound

228

to HDAH (pdb-id 2GH6) 39 and another trifluoromethylketone inhibitor bound to HDAC4 (pdb-

229

id 2VGJ) 20. In general, derivatives of trifluoromethylketones are likely promising candidates for

230

developing highly potent and selective inhibitors for this class of enzymes 43.

231

Ligand-free PA3774H143A. Based on the observed structure, several PA3774 mutants were

232

created (Table 2). The structure of the PA3774H143A mutant was solved at 1.99 Å and is

233

essentially identical with the wild type enzyme. It is worth noticing that the backbone structure

234

of the enzyme remains unchanged by the mutation and therefore the inactivation of the enzyme

235

solely results from the loss of the catalytic effect of the histidine sidechain (Fig. S9).

236

Ligand-free PA3774Y313F and complex with PFSAHA. The structure of ligand-free

237

PA3774Y313F was determined at a resolution of 2.05 Å. The structure was also solved with the

238

inhibitor PFSAHA at a resolution of 2.48 Å (Fig. 6 and S8). This inhibitor is a potent

239

perfluorinated derivative of the commercial drug SAHA (Vorinostat)

240

towards PA3774 with an IC50 value of 62 nM, whereas the affinity against human HDACs is far

241

less pronounced with IC50 values in the µM range (HDAC6: 11 µM, HDAC1: 29 µM,

242

HDAC8: 17 µM, and HDAC7: 11 µM)

243

PFSAHA, the different selectivity arises exclusively from the bulkier and polar nature of the

244

fluorine atoms. In the structure of PA3774Y313F-PFSAHA complex the hydroxamate group

44

28

and is highly selective

. Given the high similarity between SAHA and


11

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40

245

chelates the zinc ion in the typical manner as observed for HDAH (pdb-id: 1ZZ1)

246

(pdb-id: 1C3S)

247

hydroxylamine oxygen makes a hydrogen bond with H143. The catalytically active Y313 usually

248

donates a hydrogen bond to the carbonyl oxygen of the zinc chelating group adopting an

249

“inward” orientation. Yet, in the PA3774 mutant structure (Fig. 6), this Y313 is replaced by

250

phenylalanine which takes an alternative “outward” conformation pointing away from the active

251

site. Notably, in the same mutant without inhibitor, both conformations – “inward” and

252

“outward” – were found indicating the exceptional malleability of the phenyl ring at this

253

location. The reason why the “outward” conformation was exclusively observed in the inhibitor

254

bound structure might be due to the bulkier nature of the fluorine atoms and a likely repulsion of

255

the aromatic ring of F313 by partially negatively charged fluorine atoms. Moreover, the

256

“outward” conformation is in agreement with the structures of HDAC4

257

both belong to class IIa HDACs

258

from the active site. Furthermore, the “outward” conformation was found in native APAH from

259

M. ramosa (pdb-id 3Q9F)

260

warhead and the perfluorinated linker of PFSAHA is reduced (Fig. S8). However, the X-ray data

261

unequivocally prove the presence of the inhibitor within the binding pocket. The additional

262

electron density inside the binding pocket of the protein-ligand complex is not present in the free

263

mutant structure. The refined complex structure reveals a good agreement with the electron

264

density in respect to the resolution of 2.48 Å. The observed reduced electron is probably caused

265

by an incomplete occupancy of only about 70% bound PFSAHA as well as electron-withdrawing

266

effects of the adjacent fluorine atoms and the hydroxamate group on the other side.

36

or HDLP

. The hydroxylamine nitrogen forms a hydrogen bond with His144, while the

13

21

20

and HDAC7 which

. There, the corresponding histidine residue also points away

. The electron density between the Zn2+ chelating hydroxamate


12

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267

Biochemistry

Comparison with related structures.

268

The most related protein to PA3774 is HDAH from Bordetella/Alcaligenes with a sequence

269

identity of 49% (Fig. 2 and 3). The crystal structure of HDAH has been solved before

270

shows not only a structurally highly conserved core, as expected, but also a highly similar overall

271

structure with a RMSD of 0.75 Å over 269 Cα atoms. Although the overall structure of HDAH

272

has been reported as a monomer

273

similar quaternary structure as PA3774. Distinct structural differences between the two closely

274

related proteins appear in the entrance of the active site. In HDAH a “closed state” in complex

275

with acetate (pdb-id 1ZZ0) and “open state” (pdb-id 1ZZ1) in complex with SAHA was

276

observed

277

pocket is accessible regardless whether a big inhibitor or a small molecule like acetate is bound

278

to the active site. Another striking difference is the fact that PA3774 is much more active on the

279

artificial Boc-Lys(TFA)-AMC substrate compared to Boc-Lys(Ac)-AMC 27, whereas the activity

280

of HDAH on both substrates in basically identical 46.

281

PA3774 exhibits 27% sequence identity and a RMSD of 1.24 Å over 201 Cα atoms with the

282

APAH from M. ramosa, another enzyme of the HDAC superfamily. The major difference

283

between PA3774 and the APAH from M. ramosa is a loop insert (residues 76-113 in APAH)

284

which is 31 amino acids longer than the corresponding loop region in PA3774. This insert is

285

responsible for a completely different quaternary structure in APAH compared to PA3774 (Fig.

286

2C). Furthermore, this loop is not found in other HDACs and seems to be a unique feature of

287

acetylpolyamine deacetylases. This is particularly interesting because PA3774 has originally

288

been annotated as a putative APAH but shows no activity against any tested acetylpolyamine 27.

289

On the contrary, P. aeruginosa contains two further putative acetylpolamine deacetylases:

40

11, 45

11

and

, a closer look at the crystal structure of HDAH reveals a

. In contrast, such a “closed state” was not observed for PA3774 and the binding


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290

PA0321 and PA1409

291

There is no structure available for PA1409 or PA0321 but sequence analyses indicate that both

292

enzymes share a similar loop insert like the APAH from M. ramosa (indicated by the red box in

293

Fig. 3). The dimer assembly of APAH might be very different in the quaternary structure but

294

both, APAH and PA3774, have in common that the respective adjacent subunit limits the access

295

to the active site, albeit in a different manner. Presumably, this is one of the key reasons for

296

known differences in substrate selectivity and recognition. Further sequence features unique to

297

APAHs are highlighted by the red boxes in Fig. 3. For instance, two remarkable differences are

298

located inside the binding pocket. Throughout all HDACs there is a highly conserved

299

phenylalanine (F153 in PA3774 nomenclature) but in the APAH from M. ramosa, as well as in

300

both APAHs (PA1409 and PA0321) from P. aeruginosa, this residue is replaced by a tyrosine.

301

This tyrosine forms a hydrogen bond with a glutamate (E17-Y168 in APAH) in the structure

302

from M. ramosa

303

varies in all other enzymes of this family. The second difference is L276 which is replaced by an

304

isoleucine in APAHs. The exact function of these distinct differences between APAHs and all

305

other enzymes of this family is not clear, since they do not undergo direct interactions with any

306

substrate in the APAH structure. However, given their high conservation inside the active site,

307

these distinctions seem to be unique features to amidohydrolases and may thus be useful for the

308

prediction and computational annotation of yet unknown APAHs.

309

The closest sequence identity (34%) among human HDACs has HDAC10 and the second

310

domain of HDAC6 which both belong to the class IIb HDACs. Interestingly, both class IIb

311

HDACs have two deacetylase domains. HDAC6 has two functional deacetylase domains,

312

whereas HDAC10 contains only one functional domain and a second, putatively non-functional

13

. These enzymes are indeed capable of metabolizing acetylpolyamines.

. The glutamate residue is also conserved in APAHs from P. aeruginosa but


14

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Biochemistry

47

313

domain. Furthermore, both are found in the cytoplasm

. Very recently the structure of both

314

catalytical domains 1 and 2 of HDAC6 has been published, by two different groups

315

expected both structures show similar domain organizations compared to PA3774.

316

Despite the fact that PA3774 exhibits only around 20% sequence identity with class Ι HDACs,

317

their α/β fold domain as well as the monovalent cation sites are highly conserved. This is

318

represented in the small RMSD of their backbone superimposition. Differences mainly appear in

319

loop regions. With class IIa HDACs the sequence identity is about 26% and the RMSD values

320

are even lower compared to class Ι. HDACs. Detailed structure comparisons and RMSD values

321

of all HDAC and PA3774 can be found in the supplement Fig. S11.

22, 23

. As

322 323

Mutational studies. Based on the observed crystal structures we generated several mutants. The

324

amino acids H143 and H144 which form hydrogen bonds with ligands were replaced by

325

nonreactive alanines. The relative activities regarding the common substrates Boc-Lys(Ac)-AMC

326

and Boc-Lys(TFA)-AMC are listed in table 2. Neither of the single histidine mutants showed

327

detectable activity with the acetylated substrate and a weak activity (about 1%) with the

328

trifluoroacetylated

329

trifluoroacetylated substrates and a weak one against acetylated substrates

330

difference between class Ι and class IIb, in regard to class IIa HDACs, is the replacement of the

331

mechanistically important tyrosine residue near the catalytic zinc ion to a histidine. Bottemley et

332

al. demonstrated that a mutation of this histidine to tyrosine leads to significantly higher activity

333

towards acetylated lysines

334

comparison, two different mutants were produced, one where Y313 is replaced by phenylalanine

335

and another where it is replaced by histidine. Both show basically no activity against the

substrate.

20

Class

IIa

HDACs

only exhibit

high

activity 20

against

. The main

, but a reduced activity using trifluoroacetylated substrates. For


15

Biochemistry

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Page 16 of 37

336

acetylated substrate which is in line with previous studies 13, 48. However, in contrast to class IIa

337

the activity of the Y313F mutant against the trifluoroacetylated substrate was not affected and

338

only slightly reduced in the Y313H mutant. This implies that the chemical activation by the

339

trifluoroacetyl group is strong enough to completely overcome the catalytic effect of the tyrosine.

340 341 342 343

DISCUSSION

344

highly conserved protein core structures is to understand how subtle structural differences e.g. at

345

the rim of the catalytic site pocket determine selectivity in molecular recognition of substrates or

346

inhibitors. HDAH from Bordetella/Alcaligene and PA3774 from P. aeruginosa are similar with

347

respect to overall sequence identity (49%) but the imposing L1-loops of both enzymes share only

348

very low sequence identity (21%). Accordingly, one could expect that the structure of the inner

349

core of the monomeric enzymes would be very similar as is the case for all Zn2+ containing

350

enzymes from HDAC family. However, it would not be possible to predict the structure and

351

orientation of the L1-loop in PA3774 with reliable precision. Although the sequence identity is

352

very low, the structural accordance of the L1-loops in HDAH and PA3774 is surprisingly high

353

(Fig. S10) and underlines the importance of its structure for assembly of subunits. While the

354

quaternary structure of PA3774 and HDAH is maintained by the L1-loop, the related APAH

355

from M. ramosa dimerizes through a different loop

356

polyamines rather than acetylated lysine residues. The different substrate specificity is supposed

357

to be largely dependent on distinct dimer arrangements defining access to the active site pocket.

358

Similarly, the physiologically occurring multi-enzyme complexes of human HDACs in different

359

compositions are believed to show different selectivity profiles than isolated enzymes 7, 49.

Impact of quaternary structure. One of the big issues with members of the HDAC family with

13

. The substrates of APAH are acetylated


16

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Biochemistry

360

Inference of the mechanism. Two types of deacetylation mechanisms have been previously

361

discussed for zinc dependent HDACs (Fig. S12). The first one is a general acid-base catalytic

362

pair mechanism of the histidine pair H143 (inner histidine), as well as the H144 (outer histidine)

363

and was originally proposed by Finnin and co-workers

364

mechanism based on DFT QM/MM studies of HDLP

365

ab initio QM/MM MD simulations on HDAC8 51 which suggest that the outer histidine acts as a

366

general base and acid while the inner histidine is stabilizing the negative charge of the oxyanion

367

intermediate. Most recently, Gantt and colleagues published a highly comprehensive study about

368

this subject 52. Their results favor the proton shuttle mechanism and are based on the fact that a

369

mutation of outer histidine decreases activity of HDAC8 more strongly than a mutation of the

370

inner histidine. In contrast, in our enzymological study on PA3774 both histidines are equally

371

important. Both single mutations of these histidines abolish activity completely against

372

acetylated lysine substrate proving the particular importance of both histidines for the enzyme

373

mechanism. Moreover, with the trifluoroacetylated substrate the effect appears to be even

374

inverted compared with class Ι HDAC8 (Table 2). There the “outer histidine mutant” is being

375

more active than the “inner histidine mutant”. This is also in line with the recently published

376

study on HDAC6, in which the corresponding “outer histidine mutant” is also more active than

377

the “inner histidine mutant”

378

based on HDAC8 and HDLP which are both as class Ι HDACs, whereas PA3774 and HDAC6

379

are classified as class IIb HDACs, the picture of a general mechanism for all HDAC classes is

380

probably oversimplified and has to be refined by further experiments and calculations.

22

50

36

. The second one is a proton shuttle

and furthermore on Born-Oppenheimer

. Since the current discussion of a general HDAC mechanism is


17

Biochemistry

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Page 18 of 37

381

One key difference between these classes is the replacement of the outer aspartate-histidine dyad

382

to asparagine-histidine dyad in class IIb HDACs. These and other structural variations may give

383

rise to the contrary observed effect of the inner and outer histidine on enzyme activity.

384 385

In vivo function of PA3774. Against the background that PA3774 is not an APAH, the in vivo

386

function is still unclear. Since HDAH from Bordetella/Alcaligenes is highly homologous to

387

PA3774, it is obvious to speculate that these enzymes have similar functions and substrates.

388

HDAH is most active towards small peptides which contain at least one basic neighboring amino

389

acid, which is in line with the acidic electrostatic surface potential at the entrance tunnel of

390

HDAH. However, the electrostatic potential of PA3774 in this area is more basic to neutral and

391

therefore the substrate might be different. The high turnover rates of PA3774 against the

392

artificial HDAC substrates lead only to the suggestion that the natural substrate is a yet

393

unidentified lysine-acetylated protein. Recent studies revealed that many proteins in P.

394

aeruginosa are acetylated including influence factors and the DNA binding protein HU 53. It has

395

been suggested that these HU proteins have similar functions in bacteria as histones in

396

eukaryotes 54. The genome database of the P. aeruginosa strain PAO1 contains at least one HU

397

called hupB (gene number PA1805)

398

might be a substrate for PA3774. Further work to identify the substrates of PA3774 within the

399

acetylome of P. aeruginosa is ongoing.

26

. It is therefore tempting to speculate that this protein

400 401


18

Page 19 of 37

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Biochemistry

402

CONCLUSION

403

This study reveals the first structure of the HDAC like amidohydrolase PA3774 from the

404

opportunistic pathogen P. aeruginosa. The distinct tetrameric quaternary structure comprising

405

dimers of “head-to-head” dimers significantly influences the accessibility of the active site and

406

represents a crucial determinant of molecular recognition and substrate selectivity. Similarly, the

407

closely related APAH from M. ramosa

408

completely different substrate selectivity. This leads us to conclude that a full comprehension of

409

the substrate selectivity of human HDACs and their bacterial homologs is only possible by a

410

detailed understanding of the respective oligomeric states and multi enzyme complexes.

411

Consequently, the composition and structure of homo- or heteromultimeric protein complexes

412

must be considered when designing selective active substances against HDACs and HDAC like

413

proteins.

13

forms a dimer through another loop resulting in

414 415 416

AUTHOR INFORMATION

417

Corresponding Author

418

Prof.

419

E-Mail: [email protected]

420

Author Contributions

421

AK produced and crystallized the protein, solved the structures, analyzed the data. TW produced

422

and tested the mutants. AK and OY collected the diffraction data, solved and analyzed the

Dr.

Franz-Josef

Meyer-Almes,

Haardring

100,

64295

Darmstadt,

Germany


19

Biochemistry

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Page 20 of 37

423

structures. FJMA conceived the project. The manuscript was written by AK and FJMA. All

424

authors have given approval to the final version of the manuscript.

425

Funding Sources

426

This work has been supported by the Deutsche Forschungsgesellschaft (grant GZ: ME 3122/2-1).

427 428

ACKNOWLEDGEMENT

429

We want to thank the beamline scientists on the PXII - X10SA at the Swiss Light Source for

430

support during data collection and Elmar Jaenicke (University Mainz) for performing and

431

analyzing the SEC/MALLS experiments. Michael Schröder is gratefully acknowledged for his

432

generous support at the lab. We also thank Javier Carrera-Casanova and Katharina van Pee for

433

their technical support during the crystallization experiments and Gregor Rolshausen

434

(Senckenberg Institute Frankfurt) for additional proof reading.

435 436

Supporting Information Available:

437

Fig. S1: Chromatograms and PAGEs of PA3774 purification steps

438 439

Fig. S2: The modified pET21a(+) vector construct, gene sequence, amino acid sequence and mutagenesis primer

440

Fig. S3: Crystals of PA3774

441

Fig. S4: Chromatogram of the analytical SEC

442

Fig. S5: MALLS experiment

443

Fig. S6: Interfaces in the quaternary structure

444

Fig. S7: Ligand electron density map of SATFMK bound to the PA3774

445

Fig. S8: Ligand electron densities maps of PFSAHA bound to the PA3774Y313F Mutant

446

Fig. S9: Close up of the PA3774H143A mutant structure


20

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Biochemistry

447

Fig. S10: L1-Loop comparison between PA3774 and HDAH

448

Fig. S11: Comparison of PA3774 with human HDACs and HDLP

449

Fig. S12: Comparison of the two discussed mechanisms for HDACs enzymes

450

Fig. S13: IC50 value determination and structure of SATFMK

451

Fig. S14: IC50 value determination and structure of PFSAHA

452

Fig. S15: Structural formula of Boc-Lys(TFA)-AMC and Boc-Lys(Ac)-AMCThis material is

453

available free of charge via the Internet at http://pubs.acs.org

454

455

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528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

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574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619

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[38] Krissinel, E., and Henrick, K. (2007) Inference of Macromolecular Assemblies from Crystalline State, Journal of molecular biology 372, 774-797. [39] Nielsen, T. K., Hildmann, C., Riester, D., Wegener, D., Schwienhorst, A., and Ficner, R. (2007) Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor, Acta crystallographica. Section F, Structural biology and crystallization communications 63, 270-273. [40] Nielsen, T. K., Hildmann, C., Dickmanns, A., Schwienhorst, A., and Ficner, R. (2005) Crystal structure of a bacterial class 2 histone deacetylase homologue, Journal of molecular biology 354, 107-120. [41] Dowling, D. P., Gantt, S. L., Gattis, S. G., Fierke, C. A., and Christianson, D. W. (2008) Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors, Biochemistry 47, 13554-13563. [42] Gantt, S. L., Joseph, C. G., and Fierke, C. A. (2010) Activation and inhibition of histone deacetylase 8 by monovalent cations, The Journal of biological chemistry 285, 60366043. [43] Micelli, C., and Rastelli, G. (2015) Histone deacetylases: structural determinants of inhibitor selectivity, Drug discovery today 20, 718-735. [44] Henkes, L. M., Haus, P., Jager, F., Ludwig, J., and Meyer-Almes, F. J. (2012) Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases, Bioorg. Med. Chem. 20, 985-995. [45] Nielsen, T. K., Hildmann, C., Riester, D., Wegener, D., Schwienhorst, A., and Ficner, R. (2007) Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor, Acta Crystallogr Sect F: Struct Biol Cryst Commun 63. [46] Riester, D., Wegener, D., Hildmann, C., and Schwienhorst, A. (2004) Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates, Biochem. Biophys. Res. Commun. 324, 1116-1123. [47] Haggarty, S. J., Koeller, K. M., Wong, J. C., Grozinger, C. M., and Schreiber, S. L. (2003) Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation, Proceedings of the National Academy of Sciences of the United States of America 100, 4389-4394. [48] Moreth, K., Riester, D., Hildmann, C., Hempel, R., Wegener, D., Schober, A., and Schwienhorst, A. (2007) An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme, The Biochemical journal 401, 659-665. [49] Micelli, C., and Rastelli, G. (2015) Histone deacetylases: structural determinants of inhibitor selectivity, Drug Discov. Today 20, 718-735. [50] Corminboeuf, C., Hu, P., Tuckerman, M. E., and Zhang, Y. (2006) Unexpected deacetylation mechanism suggested by a density functional theory QM/MM study of histone-deacetylase-like protein, Journal of the American Chemical Society 128, 45304531. [51] Wu, R., Wang, S., Zhou, N., Cao, Z., and Zhang, Y. (2010) A proton-shuttle reaction mechanism for histone deacetylase 8 and the catalytic role of metal ions, Journal of the American Chemical Society 132, 9471-9479. [52] Gantt, S. M. F., Decroos, C., Lee, M. S., Gullett, L. E., Bowman, C. M., Christianson, D. W., and Fierke, C. A. (2016) General Base-General Acid Catalysis in Human Histone Deacetylase 8, Biochemistry 55, 820-832.


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Biochemistry

[53] Ouidir, T., Cosette, P., Jouenne, T., and Hardouin, J. (2015) Proteomic profiling of lysine acetylation in pseudomonas aeruginosa reveals the diversity of acetylated proteins, Proteomics. [54] Drlica, K., and Rouviere-Yaniv, J. (1987) Histonelike proteins of bacteria, Microbiological reviews 51, 301-319. [55] Magis, C., Taly, J. F., Bussotti, G., Chang, J. M., Di Tommaso, P., Erb, I., EspinosaCarrasco, J., and Notredame, C. (2014) T-Coffee: Tree-based consistency objective function for alignment evaluation, Methods Mol Biol 1079, 117-129. [56] Eddy, S. R. (2004) Where did the BLOSUM62 alignment score matrix come from?, Nature biotechnology 22, 1035-1036.

630 631


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Biochemistry

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Table1: Data collection and refinement statistics PA3774 SATFMK

PA3774 Acetat

PA3774 H143A

PA3774 Y313F

unliganded

unliganded

unliganded

PA3774 Y313F PFSAHA

5G0Y

5G10

5G0X

5G13

5G12

5G11

P41212 81.7, 81.7, 205.2

P41212 81.7, 81.7 , 205.9

P41212

P41212

P41212

a, b, c (Å )

P41212 81.5, 81.5, 203.2

81.4, 81.4 , 204.1

81.6, 81.6, 205.2

81.8 81.8 204.9

α, β, γ (deg)

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

I/sd(I)

5.3 (2.2)

8.2 (2.1)

5.9 (2.4)

11.2 (4.8)

6.2 (2.7)

11.4 (6.9)

Wavelength (Å) Resolution range (Å) overall observations unique reflections Completeness (%)a

1.00001

0.97902

1.00002

0.97903

1.00001

1.00002

75.68 - 2.29

75.94 - 1.71

75.70 – 1.70

75.80 - 1.99

75.79 - 2.02

75.96 - 2.48

175199

578699

885648

750443

457059

288702

31717

75585

77011

48272

46280

25472

99.9 (100.0)

100.0 (100.0)

100.0 (99.9)

99.5 (94.1)

99.4 (96.8)

100.0 (100.0)

PA3774 Dataset PDB CODE Data Processing Space group

a

a

Multiplicity

5.5 (5.5)

7.7 (8.0)

11.5 (12.2)

15.5 (11.7)

9.9 (9.1)

11.3 (11.6)

0.117 (0.502)

0.062 (0.478)

0.088 (0.339)

0.044 (0.108)

0.092 (0.467)

0.050 (0.097)

Rcrystc

0.1870

0.1579

0.1343

0.1726

0.179

0.1678

Rfreed

0.2266

0.1937

0.1840

0.2172

0.224

0.2240

5953

6359

6307

5671

6122

6011

749

746

746

746

743

744

Rpimb Refinement

total atomse e

protein residues ligand moleculese

2

2

6

6

6

6

6

6

217

625

535

454

380

276

0.009

0.018

0.013

0.009

0.010

0.011

1.36

1.46

1.51

1.32

1.35

1.58

metal ionse watere Rmsd from ideal Bond lengths (Å) Bond angles (deg)

2

Ramachandran plot (%) most favoured

94.36

95.42

95.28

95.01

95.48

95.00

allowed

4.43

3.64

3.77

4.04

3.56

3.92

outliers a

1.21

0.94

0.94

0.94

0.96

1.08

b

Number in parentheses refer to the outer shell. Given the high multiplicity for the outer shells of these data sets, Rpim is a more appropriate measure of the data quality than Rmerge. c Rcryst = ∑||Fo| - |Fc|| / ∑ |Fo|, where Fo and Fc are the structure factor amplitudes from the data and the model, respectively. Intensity calculated from replicate. d Rfree is Rcryst with 5% of test set structure factors data. e Per asymmetric unit. 632 633


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

634 Table 2: Relative activities of PA3774 mutants in % Mutant

635

Boc-Lys(TFA)-AMC Boc-Lys(Ac)-AMC

Wild type

100 ± 2

100 ± 4

H143A

1.0 ± 0.1

≤ 0.05*

H144A

1.4 ± 0.1

≤ 0.05*

Y313F

98 ± 3

≤ 0.05*

Y313H

85 ± 3

≤ 0.05*

*lower limit of detection

636 637 638 639 640 641 642 643 644 645 646


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Biochemistry

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Page 28 of 37

647 648

Figure Legends

649

Fig. 1: Overall structure of PA3774 (A) Representation of one monomer in rainbow color

650

scheme, whereas red represents the C-terminus and blue the N-terminus. The enzyme adopts an

651

open α/β fold domain. Potassium ions are colored purple and the zinc ion grey. The marked L1-

652

loop region is the key factor for tetramer formation as shown in C and D. (B) A detailed

653

topology diagram with the same color coding as in A. Helices are numbered 1-14 and ß-sheets

654

are numbered a-l. The two ß-turns and the parallel 8 stranded ß-sheets are highlighted in blue

655

boxes. (C) Top view of the “head-to-head” dimer present in the asymmetric unit. One monomer

656

is colored in grey and the other in orange. The pivotal L1-loops which make extensive contacts

657

with the adjacent monomer are colored blue. (D) Tetrameric assembly inside the unit cell. The

658

color scheme of one dimer is identical to (C). The symmetry related “head-to-head” dimer is

659

colored dark grey and bright orange. The contacts between two head-to-head dimers eventually

660

forming the final tetramer are mainly mediated by L1-Loop interactions.

661 662

Fig. 2: Comparison with related structures: (A) The superimposition of PA3774 (grey) and

663

HDAH (green) shows similar tetrameric assembly. The inset shows the comparison of the L1-

664

loop which is responsible for tetramerization in more detailed. In both enzymes the L1-loop is

665

elongated compared with other enzymes of the HDAC family. The sequence identity is only 21%

666

but the orientation of the loops is very similar and consequentially the resulting assembly of

667

subunits. (B) The superimposition of PA3774 (grey) with HDAC8 (teal) shows the close

668

structural relationship between these enzymes. The main difference compared with the

669

monomeric HDAC8 is the elongated L1-Loop (highlighted in dark blue). (C) Comparison of


28

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Biochemistry

670

PA3774 “head-to-head” dimer (grey/dark-grey) and APAH dimer from M. ramosa

671

(orange/wheat): The grey and orange monomers are superimposed. A different loop insert in

672

APAH leads to entirely different assembly as indicated. Pdb-id’s used in this figure: 1ZZ0

673

(HDAH), 1T69 (HDAC8), 4ZUM (APAH)

674 675

Fig. 3: Sequence alignment of PA3774 with HDAH (the closest related structure), PA1409 and

676

PA0321 (APAHs from P. aeruginosa), APAH from M. ramosa, HDAC8 (a class Ι HDAC), the

677

second domain of HDAC6 (a class 2IIb HDAC) and the catalytic core domain of HDAC4 (a

678

class IIa HDAC). Orange dots indicate the zinc coordinating residues, green triangles indicate

679

the residues which are involved in the deacetylation process, the black box indicates the loop

680

region which is responsible for tetramer formation in PA3774 and HDAH and the red box

681

indicates the loop region which is responsible for dimer formation in APAH. The red framed

682

amino acids indicate sequence motifs which appear to be unique to APAHs. Alignment was

683

performed with T-Coffee 55, coloring indicates similarity according to BLOSUM62 56.

684 685

Fig. 4: Binding pocket of PA3774: (A) Cross section of the protein with focus on the binding

686

pocket. The figure demonstrates the crucial role of the “head-to-head” dimer assembly for

687

substrate binding. One monomer is colored in blue, the other one in orange and the inhibitor

688

SATFMK in red (stick representation). A wide area of the V-shaped pocket (orange) gets

689

covered by the adjacent monomer (blue) resulting in a substantially narrowed entrance surface.

690

(B) Anatomy of the binding pocket in stick representation. The specific amino acids which are

691

forming the binding pocket are indicated in orange. The amino acids of the adjacent subunit


29

Biochemistry

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Page 30 of 37

692

responsible for restricting the entrance area are indicated in blue. The catalytic center consist of

693

basic and polar amino acids, the binding pocket is formed mostly by nonpolar amino acids.

694 695 696

Fig. 5: Stereo view of bound ligands to the wild type enzyme. Hydrogen bonds and metal

697

coordination are shown as green and black dashed lines, respectively. Zinc and potassium ions

698

are shown as grey and purple spheres, respectively. (A) Binding of the reaction product acetate

699

to the catalytic center. The electron density map shown is a Fo-Fc map before ligand

700

incorporation, contoured at 3 σ. (B) Binding of the inhibitor SATFMK. The ketone inhibitor is

701

binding in its gem-diol form and mimics the tetrahedral transition state. (C) Structural formula of

702

the trifluoromethylketone inhibitor SATFMK.

703 704

Fig. 6: (A) Stereoview of the ligand PFSAHA bound to the Y313F mutant. The ligand is shown

705

in orange, the residues of the binding pocket in grey and the teal colored residue belong to the

706

adjacent monomer. Hydrogen bonds and metal coordination are shown as green and black

707

dashed lines, respectively. The zinc is shown as a grey sphere. The main difference between the

708

structures is the mutated residue F313 which adopts an “inward” or “outward” conformation in

709

the inhibitor-free structure (superimposed yellow residue), whereas in the inhibitor complex only

710

the outward conformation is observed. (B) Structural formula of PFSAHA.


30

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Biochemistry

Figure 1: Overall structure of PA3774 177x149mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2: Comparison with related structures 120x110mm (300 x 300 DPI)


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Biochemistry

Fig. 3: Sequence alignment 172x102mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 4: Binding pocket of PA3774 69x131mm (300 x 300 DPI)


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Biochemistry

Fig. 5: Stereo view of bound ligands to the wild type enzyme 141x106mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6: Stereo view of the ligand PFSAHA bound to the Y313F mutant 143x58mm (300 x 300 DPI)


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Biochemistry

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Crystal Structure of a Histone Deacetylase Homologue from

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