Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated...

1

2

3Q1

4

5

6789101112131415161718192021

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

Biochimica et Biophysica Acta xxx (2013) xxx–xxx

BBAMCB-57476; No. of pages: 11; 4C: 4, 5, 6, 7, 9, 10

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bba l ip

Thermodynamic and solution state NMR characterization of the bindingof secondary and conjugated bile acids to STARD5

FDanny Létourneau, Aurélien Lorin, Andrée Lefebvre, Jérôme Cabana, Pierre Lavigne, Jean-Guy LeHoux ⁎Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
O
Abbreviations: CA, cholic acid; CDCA, chenodeoxyacid; LCA, lithocholic acid; UDCA, ursodeoxycholic acidglycocholic acid; TDCA, taurodeoxycholic acid; GDCA, glclear magnetic resonance; CD, circular dichroism; ITC, isCSD, chemical shift displacement; HSQC, Heteronuclear⁎ Corresponding author. Tel.: +1 819 821 5282.

E-mail address: [email protected] (

1388-1981/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbalip.2013.07.005

Please cite this article as: D. Létourneau, econjugated bile acids to STARD5, Biochim. B

Oa b s t r a c t
a r t i c l e i n f o
22

23

24

25

26

27

28

29

30

31

32

33

Article history:Received 15 May 2013Received in revised form 4 July 2013Accepted 9 July 2013Available online xxxx

Keywords:Cholesterol metabolismBile acidSteroidogenic acute regulatory protein (StAR)Isothermal titration calorimetryCircular dichroismNMR spectroscopy

34

ED P

RSTARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in thenon-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein,we report on the biophysical and structural characterization of the binding of secondary and conjugated bileacids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and itsepimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration andmolecularmodeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to alesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids havedifferent binding modes and display different positioning within the STARD5 binding pocket. The relativeSTARD5 affinity for the different bile acids studied is: DCA N LCA N CDCA N GDCA N TDCA N CA N UDCA. TCAand GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynam-ics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellularresponse elicited by bile acids and offers many entry points to decipher its physiological role.

© 2013 Elsevier B.V. All rights reserved.

3536
T
59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

UNCO

RREC1. Introduction

Bile acid biosynthesis is themain pathway of cholesterol catabolismand consequently plays an important role in its regulation. Bile acidsconsist of a steroid backbone containing 24 carbon atoms; the backbonecan be hydroxylated at positions C3, C7 and C12 and possesses twomethyl groups at positions C10 and C13 and a lateral side-chain in posi-tion C17 with a carboxyl group in C24 (Fig. 1). Bile acids are facial am-phiphiles: the hydroxyl groups oriented in position alpha of thesteroid nucleus and the carboxyl side-chain contribute to their hydro-philic properties, whereas the two methyl groups, oriented in positionbeta, add to their hydrophobic character (Fig. 1) [1]. In human, primarybile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 1)are synthesized from hepatic cholesterol; CA is hydroxylated at posi-tions C3, C7 and C12, whereas the hydroxyl group in C12 is absent inCDCA. Conjugation of CA and CDCA to glycine or taurine in C24(Fig. 1) increases their water solubility and renders them fully ionizedat physiological pH, preventing cell diffusion or reabsorption. Conjugat-ed bile acids are transported into the biliary ductile system anddischarged into the intestinal lumen upon ingestion of a meal. Mostbile acids are reabsorbed by a highly effective uptake system in the

80

81

82

83

84

85

86

cholic acid; DCA, deoxycholic; TCA, taurocholic acid; GCA,

ycodeoxycholic acid; NMR, nu-othermal titration calorimetry;Single Quantum Coherence

J.-G. LeHoux).

rights reserved.

t al., Thermodynamic and soiophys. Acta (2013), http://dx

distal ileum [2]. Some bile acids escape from the small intestine uptaketo the colon where major modifications can occur through bacterial ac-tions including deconjugation, dehydroxylation and epimerization [3].Removal of hydroxyl group in position 7α transforms the primary bileacids CA and CDCA to secondary bile acids deoxycholic acid (DCA) andlithocholic acid (LCA), respectively. Deconjugation and dehydroxylationincrease hydrophobicity and pKa of bile acids enabling their passivereabsorption through the intestine epithelium [4]. Ursodeoxycholicacid (UDCA), another important secondary bile acid in the human bileacid pool, originates from the epimerization of the hydroxyl group inC7 of CDCA from the alpha to beta configuration [5].

Bile acids are required to facilitate the absorption of cholesterol,fat-soluble vitamins, and lipids in the intestine. Besides their role in di-gestion, bile acids have also important regulatory functions bymodulat-ing specific cellular signaling [6]. Depending on their structure, they canbe ligands to numerous membrane and nuclear receptors that regulatetheir biosynthesis and transport. For example, bile acids bind and acti-vate various nuclear receptors such as farnesoid X receptor (FXR),pregnane X receptor (PXR), Vitamin D receptor and the membraneboundG-Protein-coupled bile acid receptor TGR5 [6]. In addition to reg-ulating their own synthesis and transport, bile acids are involved in theinitiation of the adaptive response to liver disorders [7–9].

In the hepatocytes, bile acid transport is accomplished by Na+taurocholate co-transporting peptide (NTCP) and organic anion trans-port polypeptides (OATPs) [10,11]. Bile acids are then secreted intothe canaliculus through ATP-dependent bile acid excretory pump(BSEP) and multidrug resistant proteins (MRPs) [12,13]. The majorityof the secreted bile acids are reabsorbed by the intestinal epithelial

lution state NMR characterization of the binding of secondary and.doi.org/10.1016/j.bbalip.2013.07.005

http://dx.doi.org/10.1016/j.bbalip.2013.07.005

mailto:[email protected]


http://www.sciencedirect.com/science/journal/13881981


CTED P

RO

OF

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

Bile acids name R1 R2 R3 R4

Primary

Cholic (CA) OH OH OH COOH

Chenodeoxycholic (CDCA) OH OH – COOH

Secondary

Deoxycholic (DCA) OH – OH COOH

Ursodeoxycholic (UDCA) OH OH – COOH

Lithocholic (LCA) OH – – COOH

Conjugated

Taurocholic (TCA) OH OH OH CONH(CH2)2SO3H

Glycocholic (GCA) OH OH OH CONH(CH2)COOH

Taurodeoxycholic (TDCA) OH – OH CONH(CH2)2SO3H

Glycodeoxycholic (GDCA) OH – OH CONH(CH2)COOH

Alpha face (hydrophilic)

Beta face (hydrophobic)

α

α

α

α

α

α

β

α

α

α

α

α

α

α

α

α

α

α

α

α

Fig. 1. Molecular structure of common bile acids. Hydroxyl group(s) location and orientation are given for each bile acid. Cholic acid and chenodeoxycholic acid are primary bileacids. Deoxycholic acid, lithocholic acid and ursodeoxycholic acid are secondary bile acids. Glycine and taurine conjugated forms of CA and DCA are also shown.

2 D. Létourneau et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

UNCO

RREcells through the apical sodium dependent bile acid transporter (ASBT)

[14] and exit cells via the organic solute transporters (OST) at thebasolateral membrane [15,16]. The bile acid intra-cellular trafficking isthought to be mediated by the ileal bile acid binding protein (IBABP)[17,18] and the liver bile acid binding protein (LBABP) [19,20]: how-ever, very little is known about how bile acids are shuttled in other tis-sues. The bile intracellular transport from basolateral to canalicularmembrane is not understood yet, but cytoplasmic-binding proteinsare certainly involved [21,22].

STARD5, a protein involved in the non-vesicular intra-cellular trans-port of sterols [23–26], is highly expressed in Kupffer cells, renal proxi-mal tubules [25,27–29] and peripheral macrophages [28], all of whichrespond to bile acids [30–34]. STARD5 is a member of the superfamilyof proteins with a START (Steroidogenic Acute Regulatory-relatedlipid transfer) domain. START domains are conserved protein modulesof typically 210 amino acids that adopt an α/β helix-grip fold with aninternal hydrophobic cavity for ligand binding [24,35–43]. STARD5is essentially composed of a START domain and does not have anN-terminal organelle targeting sequence [29]. Accordingly, the cellulardistribution of STARD5 in different cell types is very diverse. In humanTHP-1macrophages, STARD5 localizes mainly to the perinuclear regionand colocalizes with the Golgi [28]. In 3T3-L1 cells, STARD5 is localizedin the nucleus, and uponER stress,moves to the cytosol andmembranes[44]. Finally, in HK-2 cells, STARD5 colocalizes with the ER andrelocalizes to perinuclear and cell periphery regions upon ER stressortreatment [27,45]. Although these studies demonstrate cell-specificrelocalization of STARD5, the physiological function(s) of STARD5 re-main(s) to be elucidated.

Please cite this article as: D. Létourneau, et al., Thermodynamic and soconjugated bile acids to STARD5, Biochim. Biophys. Acta (2013), http://dx

We have previously reported that STARD5 binds primary bileacids CA and CDCA in its internal cavity with an affinity in the105 M range, indicating the possibility of physiological relevance[46]. In the present study, using NMR, ITC and CD, we have addressedthe binding of the secondary bile acids DCA, LCA and UDCA and theconjugated forms of DCA and CA to STARD5. We have found thatSTARD5 affinity is stronger for DCA and LCA than for primary bileacids. The relative affinity order of STARD5 for these molecules isas follows: DCA N LCA N CDCA N GDCA N TDCA N CA N UDCA N TCAand GCA. The thermodynamics of the binding and the location of sec-ondary bile acids inside the cavity differ from those of primary bileacids. Moreover, we have identified structural determinants that de-fine the specificity of STARD5 towards its potential natural ligands.This study represents an original and initial step to define the func-tions of STARD5.

2. Experimental

2.1. Cloning, expression and purification

The cDNA for the human STARD5 was generously provided by theStructural Genomics Consortium (Karolinska Institutet, Stockholm,Sweden). The construct was modified to remove the N-terminal TEVprotease cleavage site and to add a hexahistidine tag at the C-terminus.This new constructwas sequenced and cloned into the expression vectorpET-3a (Novagen). For 15N- or 13C, 15N-double labeling, 15N ammoniumchloride (1 g/l) and 13C glucose (3 g/l) (Cambridge Isotopes) as the solenitrogen and carbon sources were used. Escherichia coli BL21(DE3) were



T

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181182183184185186187188189190191192

193194195196

197198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217218219220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

3D. Létourneau et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

UNCO

RREC

transformed with the plasmid, grown at room temperature (23 °C)in M9 medium (100% H2O or 20% H2O: 80% D2O) and induced with1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) when OD600reached 0.6. After induction, cells were incubated for an additional18 h at room temperature prior to harvesting by centrifugation. Cellswere then resuspended in lysis buffer (3 ml/g of pellets; buffer composi-tion: 50 mMK-Phosphate, 500 mMKCl, 10 mM imidazole, pH 7.4)with2 mM TCEP, protease inhibitors (complete Mini EDTA-free inhibitorsfrom Roche), 1 mM PMSF and frozen at −80 °C. Bacterial pellets werelysed by thawing at 37 °C followed by addition of lysozyme (2 mg/ml)and DNAse (50 μg/ml). The cell lysate was then centrifuged at 19,000 gfor 30 min and the supernatant was loaded onto a Ni-NTA column(Qiagen) during 2 h at room temperature. The resin was washed twicewith lysis buffer and the STARD5 recombinant protein was eluted withelution buffer (50 mM K-Phosphate, 500 mM KCl, 250 mM imidazole,2 mM TCEP, pH 7.4). The buffer was exchanged and the protein concen-trated using Millipore UltraCel ultracentrifugation filters (10,000 DaMWCO; Amicon Canada) device into NMR buffer (50 mM K-Phosphate,50 mM KCl, 2 mM TCEP, pH 7.4) complemented with 10% D2O and0.01 mM NaN3. The final concentrations of the NMR samples were be-tween 0.8 and 1.2 mM. The identity and integrity of the final proteinsample were confirmed by SDS-PAGE (see Supplementary material,Fig. S1).

2.2. CD spectropolarimetry

Circular dichroism (CD) measurements were performed on a JascoJ-810 spectropolarimeter equipped with a Peltier-type thermostat.Routine calibration of the instrument was done with an aqueous solu-tion of d-10-(+)-camphor-sulfonic acid at 290.5 nm. Experimentswere performed using quartz cells with a path-length of 1.0 mm.For CD spectra and temperature-denaturation measurements ofSTARD5, the protein was dissolved in 10 mM phosphate at pH 7.4,to a final concentration of 10 μM. The protein concentration was de-termined spectrophotometrically at 280 nm using an extinction coef-ficient of 30,940 M−1 cm−1. The CD spectra presented are the resultsof the accumulation of ten scans at 0.1 nm intervals. Scan speeds andtime constants were chosen to allow sufficient response time andachieve favorable signal-to-noise ratios. Temperature-induced dena-turation curves were performed in the temperature range from 5 °Cto 95 °C with a rate heating of 1 °C/min. The raw mdeg values weretransformed in mean residue molar ellipticity (deg·cm2·dmol−1)using the following equation (Eq. (1)):

Θ½ �222 ¼ CD signal degð Þ �MRWconcentration g=Lð Þ � l � 10 ð1Þ

where MRW is the mean residue weight and l is the path length of theCD cell in cm. The determination of the apparent melting temperature(T°), the enthalpy of unfolding at T° (ΔH°u (T°)) and the temperaturedependent Gibbs free energy of unfolding (ΔG°u (T°)) was performedby the simulation of the temperature denaturation curves with amodel describing the equilibrium a two-state unfolding mechanismand assuming a ΔCp,u of 1 kcal·mol−1·K−1 as described in Roostaeeet al. [47]. The temperature dependent population of the unfoldedstate (Pu (T)) was obtained from the simple two-state model, wherethe folded state of STARD5 is in equilibrium with its unfolded state(Eq. (2)):

Pu Tð Þ ¼ exp −ΔG�u Tð Þ=RTð Þ

1þ expð−ΔG�u Tð Þ=RTð Þ ð2Þ

where R is the universal gas constant. The far-UV CD spectra and tem-perature denaturation were recorded at a protein to ligand molarratio of 1:100 in the NMR buffer. At such a ratio all the proteins are


ED P

RO

OF

bound by a ligand to give a maximum amount of complex. Each spec-trum was baseline-corrected for buffer and ligand.

2.3. Nuclear Magnetic Resonance (NMR) spectroscopy

NMR experiments were performed at 298 K on a Varian (Agilent)600 MHz spectrometer equippedwith a Z-axis pulsed-field gradient tri-ple resonance probe. The sequence-specific assignments of 1HN, 13Cα,13C′, 15N and side chain 13Cβ for the STARD5-DCA complexes wereobtained using 1H–15N HSQC and standard triple-resonance NMR ex-periments (e.g. HNCACB, HNCO, HN(CO)CA). NMR data were processedusing NMRPipe [48] and analyzed with CCPNmr Analysis [49]. Theassignments of STARD5 in complex with DCA are deposited in theBioMagResBank (http://www.bmrb.wisc.edu/) with accession numberpending. The spectra were referenced as described [50]. The chemicalshift values of 15N, 13Cα and 13Cβ have been corrected for the deuteriumisotopic effect using the values described in Gardner et al. [51]. The as-signment of the backbone chemical shifts allows the identification ofthe backbone amide cross-peaks significantly shifted by the presenceof ligands. From those assignments, chemical shift displacement (CSD)profiles can be established. The CSD is calculated with the followingequation (Eq. (3)):

CSD ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiΔ1H

� �2 þ Δ15N= SW1H=SW15N� �� 2

� �

2

vuuut ð3Þ

where SW is the spectral width of both dimensions. Significant dis-placements are attributed to cross-peaks that have CSD values one SDabove the mean.

2.4. Isothermal titration calorimetry (ITC)

ITC measurements were performed using a VP-ITC (GE Healthcare-MicroCal, Northampton, MA) at 25 °C. Protein concentration was cho-sen in order to have a c value (i.e. the product of the binding constant(Ka), the protein concentration ([P]) and the stoichiometric parameter(n)) between 10 and 100 and a peak height of more than 0.5 μcal/s.The ligand concentrations used were below their respective critical mi-celle concentration values. Each experiment consisted of 49 injectionsinto the experimental chamber of 6 μl of the ligand solution at 3 min in-tervals at a stirring speed of 260 rpm. Heats of dilution were subtractedfrom the raw titration data before analysis. Experiments were per-formed in triplicate and data were fitted by least-square proceduresassuming a one-site binding model using Microcal Origin version 7.0.

2.5. Molecular modelling and molecular dynamics simulations

Coordinates for the missing residues in the α3 helix of the crystalstructure of STARD5 (2R55; [42]) were generated by introducingthem in the model in an α-helical conformation as previously de-scribed [52]. Then, the potential energy in that region was minimizedwhile restraining the rest of the molecule [46]. The GROMACS soft-ware suite [53–56] was used to prepare and run the simulations.The STARD5 protein was solvated with the SPC water model [57]and approximately 50 mMNaCl, keeping the net charge of the systemat 0. The STARD5 protein was centered in a dodecahedric box in peri-odic boundary conditions with each side of the box being at least 10 Åfrom the protein to prevent any interaction between the periodic im-ages of STARD5. The Gromos 53a6 force field was used for the calcu-lations [58,59]. The DCA molecule was created and parameterized forthe force field by using the cholate molecule parameters available onthe Automated Topology Builder repository [60]. The DCA moleculewas docked in the binding site of STARD5 as described in Letourneauet al. [46]. Equilibration of the system under conditions of constantnumber of molecules, volume and temperature (NVT) was performed


http://www.bmrb.wisc.edu/


253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293


for 50 ps to reach the desired temperature of 310 K. This was follow-ed by equilibration under conditions of constant number of mole-cules, pressure and temperature (NPT) for 100 ps with the pressureset at 1 bar. Unrestrained MD simulations were run for 40 ns in 2 fssteps. The simulations were run at constant temperature (310 K) andpressure (1 bar) using the velocity-rescaling thermostat [61] withτT = 0.2 ps and the Parrinello–Rahman barostat with τP = 5 ps, re-spectively. Simulation data were saved every 2 ps, for a total of 20,000frames. MD trajectory output from GROMACS was converted to PDBfiles with 100 frames (1 for every 200 saved) for visual inspectionwith PyMOL (The PyMOL Molecular Graphics System, Version 1.3r1Schrödinger, LLC).

3. Results

3.1. Monitoring of the binding of secondary bile acids to STARD5 bysolution-state NMR

The 1H–15N HSQC spectra of STARD5 in the absence and presence ofthe DCA, LCA and UDCA are shown in Fig. 2A, B and C, respectively. Theaddition of 2 equivalents of these bile acids to STARD5 induces the dis-placement of numerous cross-peaks (commonly referred to as chemical

UNCO

RRECT

Fig. 2. Secondary bile acids binding to STARD5. Overlain of 1H–15N HSQC spectra of STARD5the bile acid. (A) STARD5-DCA complexes (red). (B) STARD5-LCA complexes (red). (C)STARD5-DCA spectra (red).


OO

F

shift displacements or CSDs) on the 1H–15N–HSQC of STARD5. More-over, the addition of secondary bile acids causes the equivalent cross-peak displacement on STARD5 HSQCs, indicating that a single subsetof residues is interacting with them and confirming the existence of acommon binding site. The residue identity and the extent of the CSDsinduced by secondary bile acids differ from those of the primary bileacid CA [46] (Fig. 2D). This suggests a different mode of interactionand/or the induction of a conformational change of the protein uponbinding of secondary bile acids.

To assess the origin of these differences in CSDs, we have reassignedthe backbone chemical shifts of STARD5 (BMRB access, pending) in thepresence of DCA (Fig. 3A and B). This was done to unambiguously iden-tify the backbone amides of all the residues of the bound form of theprotein and to verify if notable conformational changes occur uponbinding of DCA. Fig. 3C and D depicts the secondary chemical shifts forthe Cα, Cβ, C′ and the CSI as a function of the primary and secondarystructures for the apo-STARD5 and STARD5-DCA complex. Briefly, thesecondary chemical shifts of backbone nuclei (Cα, Cβ, C′) of a residueindicates whether it is in α-helical or β-sheet conformation. Positivesecondary chemical shifts for Cα and C′ and negative secondary chem-ical shifts for Cβ indicate that a residue is inα-helical configuration. Fora residue in β-sheet configuration these relationships are reversed. The

ED P

R

(0.8 mM in NMR buffer at pH 7.4) alone (black) and in the presence of 2 equivalents ofSTARD5-UDCA complexes (red). (D). Comparison between STARD5-CA (green) and



294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326Q2327

328

329

330


CSI is a consensus score obtained from the δΔ values [62]. Stretches ofpositive and negative CSIs are indicative of α-helices and β-strands, re-spectively. The presence of DCA does not modify the secondary struc-ture of apo-STARD5 (Fig. 3C and D). Hence, the difference in CSDsindicates a different mode of interaction of secondary bile acids com-pared to primary bile acids.

Shown in Fig. 4A is a schematic representation of the CSDsrecorded for STARD5 in the presence of CA and DCA. The CSDs in-duced by DCA are located further away from α2- and α3-helices,suggesting that the location of DCA inside the binding cavity is differ-ent. As shown in Fig. 4B, the CSDs for LCA, which also lacks the α-OHat C7 (Fig. 1), are highly similar to that of DCA. This suggests that theα-OH at C7 is critical for the positioning of primary and secondarybile acids inside STARD5. Interestingly, the CSD pattern of UDCA,which has a β-OH at C7 (Fig. 1), is similar to those of DCA and LCA(Fig. 4A–B), confirming the role of the α-OH at C7 for the positioningof bile acids in the internal cavity of STARD5. This signifies that DCA,LCA and UDCA bind at a different location than the primary bileacids, as illustrated in Fig. 4E and F for CA and DCA, respectively.

UNCO

RRECT

Fig. 3. Backbone chemical shift assignments of STARD5-DCA complexes. (A) Overlain of 1H–1

presence of 2 equivalents of DCA (red). Assignments of the cross-peaks are given with thside-chain resonances of asparagines and glutamines. Tryptophan side-chain indole NH crguanidino (Hη) protons from arginine side-chains. (B) Detailed view of the congested reshift index) and secondary structure profile from CSI values for apo-STARD5. α-Helices aSame as in (C) but for STARD5-DCA complexes.


OF

3.2. Thermodynamic analysis of the binding of secondary bile acidsto STARD5

Using CD, we have measured the effect of the secondary bile acidson the thermodynamic stability of STARD5. There is a direct relation-ship between the stabilization free energy provided by the binding ofa ligand (estimated by the increased in the melting temperature; ΔT°)and the apparent affinity (Ka) [63]. As for primary bile acids [46] andin accordance with our NMR results (Fig. 3A–B), no changes in thefar-UV CD spectrum and secondary structure content are observedfor STARD5 in the presence of DCA, LCA or UDCA (Fig. 5A). However,the T° of STARD5 is significantly increased by secondary bile acids(Fig. 5B–C), as expected for the favorable binding of a ligand. The sim-ulation of STARD5 temperature denaturation with a two-state model(see Materials and methods) is shown in Fig. 5B and E (black). Fig. 5Cand F (black) illustrates the corresponding population of the unfoldedstate (Pu) vs. Temperature. From this simulation and Gibbs–Helm-holtz equation, apparent values for T° and ΔH°u (T°) were determinedand ΔG°u (25 °C) and ΔG°u (37 °C) values were calculated (Table 1).

ED P

RO

5N HSQC spectra of STARD5 (0.8 mM in NMR buffer at pH 7.4) alone (black) and in thee residue number and one-letter code for amino acids. Horizontal lines connect theoss-peaks are marked with an asterisk (*). The double asterisks (**) denote protectedgion of the spectrum. (C) Secondary chemical shifts of 13Cα, 13Cβ, 13C′, CSI (chemicalnd β-strands are identified by consecutive CSI values of −1 and 1, respectively. (D)



TED P

RO

OF

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

Fig. 4.Mapping the STARD5 binding site for different bile acids. Weighted chemical shift displacements (CSD) map upon binding to STARD5 of (A) CA or DCA. (B) UDCA or LCA. (C)GDCA or TDCA. (D) Comparison between DCA and GDCA. Dashed line represented the mean value of CSD for each ligand. (E) Normalized CSDs mapped onto STARD5 structure forCA and (F) for DCA.


UNCO

RRECSTARD5 is stably folded at both temperatures and the apparent T° is

significantly increased in presence of secondary bile acids (Fig. 3Band C); this indicates that the stabilization free energy provided bybile acid binding is favorable. Since there is a direct relationship be-tween ΔT° induced by ligand binding and their actual Ka [63], the af-finity (or Ka) follows this order: DCA N LCA N UDCA. Combining theseresults and those previously reported for primary bile acids [46], therelative apparent affinity order of the bile acids tested so far forSTARD5 is: DCA N LCA N CDCA N CA N UDCA.

Fig. 6A–C shows the binding isotherms for the titration of STARD5with UDCA, LCA and DCA, respectively. The thermodynamic parame-ters obtained from the fits are compiled in Table 2. Contrary to thebinding of the primary bile acids (Fig. 6D) [46], which was entropydriven, the binding reaction of the secondary bile acids to STARD5 isexothermic (negative enthalpy of binding (ΔHa)). This indicates thatthe binding free energy is enthalpy driven; DCA, LCA and UDCAmakemore favorable interactions with STARD5 than with the solvent.The corresponding Ka values (Table 2) also confirms the ranking in af-finity obtained for the secondary bile acids with the thermal denatur-ation monitored by CD.

3.3. Binding of conjugated CA and DCA to STARD5

Since many bile acid carriers/transporters specifically bind theconjugated form of bile acids, we have addressed the possibility thatSTARD5 could bind the conjugated forms of CA and DCA (Fig. 1).The 1H–15N HSQC spectra of STARD5 were recorded in the presenceof TCA, GCA, TDCA and GDCA (molar ratio of STARD5:ligand; 1:2).As for unconjugated bile acids, no apparent changes in secondarystructure composition are observed by CD for GCA, TCA, TDCA andGDCA (Fig. 5D). The 1H–15N HSQC spectra of STARD5 (Fig. 7A–B)


show that GCA and TCA induce minor or no significant chemicalshift displacements compared to the binding of their unconjugatedcounterpart CA (Fig. 2D). This indicates that the binding of TCA andGCA is very weak and does not provide enough free energy of stabili-zation for the formation of a significant amount of complex. Thislower affinity is confirmed by CD denaturation (Fig. 5E–F) and ITCcurves (Fig. 6E–F). Actually, the ΔT° induced by TCA and GCA issmall (Table 1). Moreover, as expected from the lower cooperativityof the denaturation curves, the apparent ΔH°u (T°) determined bythe fitting is smaller than the apo-STARD5. Consequently, the appar-ent ΔG°u (25–37 °C) in the presence of both ligands is lower thanthe one of the apo-STARD5. This strongly suggests that both conjugat-ed forms of CA do not bind specifically to STARD5 and probably stabi-lize an intermediate form of the START domain (Table 1) [46].Similarly, ITC measurements of STARD5 with TCA and GCA led toill-defined Kas and ΔHas (Table 2). Taken together, these results indi-cate that GCA and TCA are not STARD5 ligands and measured affini-ties are not specific.

The binding affinities of the conjugated forms of DCA (TDCA andGDCA) are comparable to those of UDCA (Table 2), CA and CDCA[46]. In fact, on the one hand, the 1H–15N HSQC spectra and the CSDpatterns observed for GDCA and TDCA are reminiscent of DCA(Fig. 7C–D and Fig. 4C–D). On the other hand, the stability of theSTARD5 in complex with TDCA or GDCA is even higher than what isdetermined for UDCA (Fig. 5C and F and Table 1). Peculiarly, the dena-turation curve of STARD5 in the presence of TDCA is significantly lesscooperative. Such a phenomenon could be induced by the stabiliza-tion of an intermediate state. The Ka values obtained for TDCA andGDCA are 30 and 20 fold less than that measured for DCA and approx-imately 2 to 3-fold higher than the Ka of UDCA, respectively (Table 2).However, compared to DCA, the presence of the charged tauryl and



TD P

RO

OF

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

t1:1

t1:2

t1:3

t1:4

t1:5

t1:6

t1:7

t1:8

t1:9

t1:10

t1:11

t1:12

t1:13

t1:14

Fig. 5. Far-UV CD spectroscopy of STARD5. (A and D) Mean residue ellipticity of STARD5 bound with secondary and conjugated bile acids: black curve, apo-STARD5. (B and E) Ther-mal melting curve recorded at 222 nm. (C and F) Temperature-dependence of the population of unfolded state (Pu). The concentrations of STARD5 and bile acids are 10 μMand 1mM, respectively. Considering Ka values in the 104 M−1 range it can be calculated that the population of complexes at 25 °C is 100% with such concentrations of STARD5and ligands.


glycyl moieties leads to endothermic ΔHa (Fig. 6G–H). As discussedbelow, this may be the result of an incomplete compensation of theenthalpy of dehydration of the charged tauryl and glycyl moieties incomplex with STARD5.
C 412
413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

CO

RRE3.4. Binding site localization of secondary bile acids in STARD5 by NMR

We have proceeded to a molecular dynamics simulation of theSTARD5-DCA complex to identify stable configurations that wouldbe in accordance with the CSD data and that could explain the differ-ences in the sign of the enthalpy. From the simple inspection of theCSDs in Fig. 4A, it is evident that DCA interacts significantly moreclosely with residues 82–84, 100–104, 114–115, 153–154 and 165and does not appear to interact as closely, compared to primary bileacids [46], with residues 158 and 178. We have previously establishedthat for primary bile acids (CA and CDCA), the side-chain of Cys158 iswithin van der Waals contact of C1 and C2 and the side-chain OH ofThr178 is donating an H-bond to the carboxylate [46]. Also, theα-OH at position C7 is engaged in an H-bond with the OH of the

UN 428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

Table 1Thermodynamic stability of apo-STARD5 and STARD5 bile acid complexes obtainedfrom the simulation of the temperature denaturation.

T° (°C) ΔH°u (T°)a ΔG°u (25 °C) ΔG°u (37 °C)

D5 (apo) 47.5 (±0.2)b 146.2 (±3.7)b 9.4 (±0.5) 4.6 (±0.5)UDCA 50.9 (±0.3) 137.0 (±4.1) 9.9 (±0.6) 5.6 (±0.6)DCA 58.0 (±0.4) 119.7 (±3.7) 10.2 (±0.8) 6.9 (±0.8)LCA 56.9 (±0.4) 126.1 (±4.3) 10.6 (±0.8) 7.0 (±0.8)GCA 49.4 (±0.7) 92.8 (±7.1) 6.1 (±1.5) 3.3 (±1.5)TCA 50.0 (±0.4) 111.4 (±3.2) 7.6 (±0.7) 4.2 (±0.7)GDCA 55.2 (±0.5) 129.1 (±6.7) 10.4 (±1.1) 6.6 (±1.1)TDCA 55.1 (±0.7) 124.1 (±3.5) 10.0 (±1.3) 6.3 (±1.3)

a Values given in kcal·mol−1.b Standard deviation of the fits.


Eside-chain of Thr101 and, finally, Thr103 is participating to the inter-nal solvation of the carboxylate [46].

Fig. 8A displays a frame trajectory of molecular dynamics simula-tion of STARD5-DCA complex in which the ligand position is in accor-dance with CSD changes. Theα-OH absence at C7 allows DCA to moveaway from residues 158 and 178. Compared with the poses obtainedwith the primary bile acids (CA and CDCA) [46], one can notice thatthe carboxylate of DCA is retaining an H-bond with Thr103 but nowinteracts (via an H-bond and electrostatically) with the guanidinogroup of Arg114 rather than the side-chain of Thr178. However, theinteraction of the side-chain OH of Ser132 with the C3 hydroxyl ismaintained and contributes to hydrogen bonding network with DCA(Fig. 8A). From the CSDs (Fig. 4A), Val83 shows stronger perturbationwith DCA than with CA, perhaps due to hydrophobic interactionswith the steroid backbone. Also, the salt bridge between Arg76 andAsp80 seems less affected by the presence of DCA and this can beexplained by the absence of interaction with the ligand (Figs. 4Aand 8A). As suggested, the phenylalanine residues (Phe116, Phe176,Phe193 and Phe200) must impose spatial constraints inside the cavityand contribute to the ligand specificity [41].

To evaluate whether the β-OH and α-OH at C7 affect differentlysome STARD5 regions, CSD data for UDCA and LCA were compared;residues 82–84 in strand β4 and 68–69 in helix α3 are significantlymore disturbed by UDCA (Figs. 4B and 8C). The presence of theβ-OH to one side of the cavity must repel the ligand in the oppositedirection and leads to more perturbations in this region. Similarly,the presence of a longer lateral chain, as for the conjugated GDCA,should cause differences in CSD when compared to DCA (Fig. 4D). In-deed, the glycyl moieties appear to disturb a remote area of the cavity,around Asp115, Arg150 and Asn153 (Fig. 8D). The very weak bindingof GCA and TCA does not allow accurate calculation of chemical shiftdisplacements. In all cases, the residue Gly165, which is remote fromthe cavity of STARD5, shows substantial CSD that must be the result ofan indirect and long range perturbation originating from the ligandbinding (Fig. 4A–F).



CTED P

RO

OF

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

Fig. 6. Determination of the Ka of bile acids for STARD5. Isothermal titration calorimetry (ITC) measurements of STARD5 with (A) UDCA. (B) LCA. (C) DCA. (D) CA. (E) GCA. (F) TCA.(G) GDCA and (H) TDCA. Raw data at 25 °C (upper panels) and integrated heat changes (bottom panels), corrected for the heat of dilution and fitted to a single site model.

t2:1

t2:2

t2:3

t2:4

t2:5

t2:6

t2:7

t2:8

t2:9

t2:10

t2:11

t2:12

t2:13

t2:14

t2:15


CO

RREFinally, as discussed in Letourneau et al. [46], the enthalpic cost of

desolvating a carboxylate is high. We have therefore proposed thatthe positive enthalpy measured for the binding of CA and CDCAcame for an incomplete compensation of the desolvation of the car-boxylate by the two H-bonds with Thr103 and Thr178. However,our CSD data clearly suggest the existence of an interaction betweenDCA (LCA and UDCA) with Arg114. Enthalpically, the interactionbetween the guanidino and the carboxylate is expected to be morefavorable leading to stronger hydrogen bonds (more important de-crease in enthalpy), which may well explain the change in sign ofthe ΔHa.

UN

Table 2Thermodynamic parameters for the binding of secondary and conjugated bile acids to STAR

Kaa ΔHa

b

Secondary bile acidsDCA 103 (±9) −5.24 (±0.30)LCA 50.0 (±11.8) −6.97 (±0.26)UDCA 1.39 (±0.20) −4.47 (±0.71)

Conjugated bile acidsTCA 0.68 (±0.06) 1.22 (±0.06)GCA 0.06 (±0.02) 385 (±435)TDCA 3.33 (±0.20) 7.53 (±0.15)GDCA 5.18 (±0.48) 6.38 (±0.17)

a ·104 M−1.b Values given in kcal·mol−1.


4. Discussion

To gain further insight into the function of STARD5, we have studiedits selectivity towards secondary and conjugated bile acids using CD,NMR and ITC.We have clearly established that STARD5 binds the second-ary bile acids DCA (Ka = 1.03 ∙ 106 M−1), LCA (Ka = 0.50 ∙ 106 M−1)and UDCA (Ka = 0.01 ∙ 106 M−1) as well as the conjugated forms ofDCA, TDCA (Ka = 0.03 ∙ 106 M−1) and GDCA (Ka = 0.05 ∙ 106 M−1).We have determined that the conjugated forms of DCA bind to STARD5with similar affinities than that previously reported for CA and CDCA[46]. Worth mentioning, the affinities of STARD5 for all the above bile

D5 determined by ITC at 25 °C.

T ∙ ΔS°ab ΔG°ab n

2.96 (±0.73) −8.20 (±0.70) 1.01 (±0.01)0.80 (±2.09) −7.77 (±1.83) 0.86 (±0.02)1.18 (±1.40) −5.65 (±0.69) 0.82 (±0.10)

6.45 (±0.54) −5.23 (±0.48) 1.94 (±0.07)390 (±437) −3.75 (±1.25) 0.03 (±3.14)13.7 (±0.53) −6.17 (±0.38) 0.76 (±0.02)12.8 (±0.77) −6.43 (±0.60) 0.85 (±0.02)



RRECTED P

RO

OF

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

Fig. 7. Conjugated bile acids binding to STARD5. Overlain of 1H–15N HSQC spectra of STARD5 (0.8 mM in NMR buffer at pH 7.4) alone (black) and in the presence of 2 equivalentsof the conjugated bile acid. (A) STARD5-GCA complexes (purple). (B) STARD5-TCA complexes (purple). (C) STARD5-GDCA complexes (blue). (D) STARD5-TDCA complexes(blue).


UNCO

acids lie in the range of physiological concentrations [64]. Interestingly, incontrast to the conjugated forms of DCA, the conjugated forms of CA (TCAand GCA) do not present enough affinity to provide significant complexformation and hence, must not be considered as specific STARD5 ligands.

By combining these results and those previously reported for theprimary bile acids [46], we have determined that the order of relativeaffinities of the bile acids tested for STARD5 is: DCA N LCA N CDCA N

GDCA N TDCA N CA N UDCA N TCA and GCA. These affinities are in thesame order of magnitude as that of other bile acid binding proteins.For example, the bile acid rank of potency for TGR5 is LCA N DCA N

CA N UDCA; conjugationwith taurinemoiety slightly increases the ago-nist potency to TGR5whereas conjugationwith glycinemoiety has onlya negligible effect on TGR5 activity [65]. Additionally, the affinity ofSTARD5 for primary bile acids is similar to that reported for FXR [32].By comparison, the binding affinity to IBABP is in the same concentra-tion range as STARD5 [66,67] and IBABP also binds UDCA [5]. Analo-gously to STARD5, IBABP ligand recognition is determined by thepattern of steroid ring hydroxylation (C3, C7 and C12) and not by theside-chain conjugation (C24) [68]. ASBT and NTCP transporters bind a


wide variety of bile acids, but contrary to STARD5 all major C-7 modifi-cations resulted in analogs that did not display active uptake by bothtransporters [69].

The main structural difference between primary and secondary bileacids is the α-OH group at position C7. The removal of the C7 hydroxylfrom CA to DCA leads to an increase of STARD5 affinity more than fiftytimes. The importance of the OH group in position 7α is furtherhighlighted by the fact that its epimerization to 7β considerably lowersits affinity towards STARD5. This position is thus crucial for the ligandbinding affinity and must define largely the specificity towards the li-gand. STARD5 affinity for its ligand is less affected by changes in C12 po-sition; it halved when going from DCA to LCA (C12 α-OH withdrawal)and decreased by one third when passing from CDCA to CA (C12α-OH addition). The magnitude of the affinity depends mainly on thenumber and positions of the steroid ring hydroxyl groups and to a lesserextent on the presence or type of bile acid side-chain conjugation.

The physiological relevance of these findings remains to be investi-gated. Nevertheless, because: 1—STARD5 is highly expressed in cells re-sponsive to bile acids, 2—binds bile acids at physiological concentrations



TD P

RO

OF

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516517518519520521522523524525526527528529530531532533

534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577

Fig. 8. STARD5 ligand binding site. (A) Molecular dynamics simulation of the STARD5-DCA complex: representative and corresponding frame modeling the complex in accordancewith the CSD data. The H-bonds suggested to lead to specific binding are highlighted with dashed lines. Normalized CSD differences mapped onto STARD5 structure between (B)DCA and CA, (C) UDCA and LCA and (D) GDCA and DCA.


UNCO

RREC

and 3—relocates within cells under various conditions, one can reason-ably postulate that STARD5 is involved in the cellular responses elicitedby bile acids.

Funding

This work was supported by a grant (MT-10983) to JGL and PL fromthe Canadian Institutes of Health Research and by the RegroupementStratégique sur la Structure, la Fonction et l'Ingénierie des Protéines(PROTEO).

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbalip.2013.07.005.

Acknowledgements

We thank Dr Lari Lehtiö (Åbo Akademi University, Turku, Finland)for kindly providing us with the STARD5 cDNA.

References

[1] M.J. Monte, J.J. Marin, A. Antelo, J. Vazquez-Tato, Bile acids: chemistry, physiology,and pathophysiology, World J. Gastroenterol. 15 (2009) 804–816.

[2] S.M. Houten, J. Auwerx, The enterohepatic nuclear receptors are major regulatorsof the enterohepatic circulation of bile salts, Ann. Med. 36 (2004) 482–491.

[3] O. Bortolini, A. Medici, S. Poli, Biotransformations on steroid nucleus of bile acids,Steroids 62 (1997) 564–577.

[4] K.D. Setchell, A.M. Lawson, N. Tanida, J. Sjovall, General methods for the analysisof metabolic profiles of bile acids and related compounds in feces, J. Lipid Res. 24(1983) 1085–1100.

[5] C. Fang, F.V. Filipp, J.W. Smith, Unusual binding of ursodeoxycholic acid to ilealbile acid binding protein: role in activation of FXRalpha, J. Lipid Res. 53 (2012)664–673.

[6] P. Lefebvre, B. Cariou, F. Lien, F. Kuipers, B. Staels, Role of bile acids and bile acidreceptors in metabolic regulation, Physiol. Rev. 89 (2009) 147–191.

[7] J.Y. Chiang, Bile acid regulation of gene expression: roles of nuclear hormone re-ceptors, Endocr. Rev. 23 (2002) 443–463.

[8] J.J. Eloranta, P.J. Meier, G.A. Kullak-Ublick, Coordinate transcriptional regulation oftransport and metabolism, Methods Enzymol. 400 (2005) 511–530.


E

[9] A. Geier, M. Wagner, C.G. Dietrich, M. Trauner, Principles of hepatic organic aniontransporter regulation during cholestasis, inflammation and liver regeneration,Biochim. Biophys. Acta (BBA) - Mol. Cell Res. 1773 (2007) 283–308.

[10] B. Hagenbuch, P.J. Meier, Molecular cloning, chromosomal localization, and func-tional characterization of a human liver Na+/bile acid cotransporter, J. Clin. In-vest. 93 (1994) 1326–1331.

[11] G.A. Kullak-Ublick, M.G. Ismair, B. Stieger, L. Landmann, R. Huber, F. Pizzagalli, K.Fattinger, P.J. Meier, B. Hagenbuch, Organic anion-transporting polypeptide B(OATP-B) and its functional comparison with three other OATPs of human liver,Gastroenterology 120 (2001) 525–533.

[12] T. Gerloff, B. Stieger, B. Hagenbuch, J. Madon, L. Landmann, J. Roth, A.F. Hofmann,P.J. Meier, The sister of P-glycoprotein represents the canalicular bile salt exportpump of mammalian liver, J. Biol. Chem. 273 (1998) 10046–10050.

[13] H. Akita, H. Suzuki, K. Ito, S. Kinoshita, N. Sato, H. Takikawa, Y. Sugiyama, Charac-terization of bile acid transport mediated by multidrug resistance associated pro-tein 2 and bile salt export pump, Biochim. Biophys. Acta 1511 (2001) 7–16.

[14] A.L. Craddock, M.W. Love, R.W. Daniel, L.C. Kirby, H.C. Walters, M.H. Wong, P.A.Dawson, Expression and transport properties of the human ileal and renalsodium-dependent bile acid transporter, Am. J. Physiol. 274 (1998) G157–G169.

[15] P.A. Dawson, M. Hubbert, J. Haywood, A.L. Craddock, N. Zerangue, W.V. Christian,N. Ballatori, The heteromeric organic solute transporter alpha-beta, Ostalpha–Ostbeta, is an ileal basolateral bile acid transporter, J. Biol. Chem. 280 (2005)6960–6968.

[16] N. Ballatori, W.V. Christian, J.Y. Lee, P.A. Dawson, C.J. Soroka, J.L. Boyer, M.S.Madejczyk, N. Li, OSTalpha–OSTbeta: a major basolateral bile acid and steroid trans-porter in human intestinal, renal, and biliary epithelia, Hepatology 42 (2005)1270–1279.

[17] J.F. Landrier, J. Grober, I. Zaghini, P. Besnard, Regulation of the ileal bile acid-binding protein gene: an approach to determine its physiological function(s),Mol. Cell. Biochem. 239 (2002) 149–155.

[18] J. Grober, I. Zaghini, H. Fujii, S.A. Jones, S.A. Kliewer, T.M. Willson, T. Ono, P.Besnard, Identification of a bile acid-responsive element in the human ileal bileacid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoicacid receptor heterodimer, J. Biol. Chem. 274 (1999) 29749–29754.

[19] L. Ragona, M. Catalano, M. Luppi, D. Cicero, T. Eliseo, J. Foote, F. Fogolari, L. Zetta,H. Molinari, NMR dynamic studies suggest that allosteric activation regulatesligand binding in chicken liver bile acid-binding protein, J. Biol. Chem. 281(2006) 9697–9709.

[20] H.L. Monaco, Review: the liver bile acid-binding proteins, Biopolymers 91 (2009)1196–1202.

[21] B. Stieger, A. Geier, Genetic variations of bile salt transporters as predisposing fac-tors for drug-induced cholestasis, intrahepatic cholestasis of pregnancy and ther-apeutic response of viral hepatitis, Expert Opin. Drug Metab. Toxicol. 7 (2011)411–425.





T

578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631632633634635636637638639640641642643644

645646647648649650651652653654655656657658659660661662663664665666Q3667668669670671672673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711

713


CO

RREC

[22] L.B. Agellon, E.C. Torchia, Intracellular transport of bile acids, Biochim. Biophys.Acta 1486 (2000) 198–209.

[23] F. Alpy, C. Tomasetto, Give lipids a START: the StAR-related lipid transfer (START)domain in mammals, J. Cell Sci. 118 (2005) 2791–2801.

[24] C.P. Ponting, L. Aravind, START: a lipid-binding domain in StAR, HD-ZIP and sig-nalling proteins, Trends Biochem. Sci. 24 (1999) 130–132.

[25] D. Rodriguez-Agudo, S. Ren, P.B. Hylemon, K. Redford, R. Natarajan, A. Del Castillo,G. Gil, W.M. Pandak, Human StarD5, a cytosolic StAR-related lipid binding pro-tein, J. Lipid Res. 46 (2005) 1615–1623.

[26] J.F. Strauss III, T. Kishida, L.K. Christenson, T. Fujimoto, H. Hiroi, START domainproteins and the intracellular trafficking of cholesterol in steroidogenic cells,Mol. Cell. Endocrinol. 202 (2003) 59–65.

[27] Y.C. Chen, R.K. Meier, S. Zheng, S.J. Khundmiri, M.T. Tseng, E.D. Lederer, P.N.Epstein, B.J. Clark, Steroidogenic acute regulatory-related lipid transfer domainprotein 5 localization and regulation in renal tubules, Am. J. Physiol. Renal Phys-iol. 297 (2009) F380–F388.

[28] D. Rodriguez-Agudo, S. Ren, P.B. Hylemon, R. Montanez, K. Redford, R. Natarajan,M.A. Medina, G. Gil, W.M. Pandak, Localization of StarD5 cholesterol binding pro-tein, J. Lipid Res. 47 (2006) 1168–1175.

[29] R.E. Soccio, R.M. Adams, M.J. Romanowski, E. Sehayek, S.K. Burley, J.L. Breslow,The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer pro-tein with two closely related homologues, StarD5 and StarD6, Proc. Natl. Acad.Sci. U. S. A. 99 (2002) 6943–6948.

[30] J.H. Miyake, S.L. Wang, R.A. Davis, Bile acid induction of cytokine expression bymac-rophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase,J. Biol. Chem. 275 (2000) 21805–21808.

[31] R.A. Davis, Resolving the mechanism of bile acid negative-feedback regulation, aJournal of Lipid Research tradition, J. Lipid Res. 49 (2008) 2–3.

[32] A. Mencarelli, B. Renga, E. Distrutti, S. Fiorucci, Antiatherosclerotic effect offarnesoid X receptor, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H272–H281.

[33] T.W. Pols, M. Nomura, T. Harach, G. Lo Sasso, M.H. Oosterveer, C. Thomas, G. Rizzo,A. Gioiello, L. Adorini, R. Pellicciari, J. Auwerx, K. Schoonjans, TGR5 activation in-hibits atherosclerosis by reducing macrophage inflammation and lipid loading,Cell Metab. 14 (2011) 747–757.

[34] H. Lee, Y. Zhang, F.Y. Lee, S.F. Nelson, F.J. Gonzalez, P.A. Edwards, FXR regulates or-ganic solute transporters alpha and beta in the adrenal gland, kidney, and intes-tine, J. Lipid Res. 47 (2006) 201–214.

[35] L.M. Iyer, E.V. Koonin, L. Aravind, Adaptations of the helix-grip fold for ligandbinding and catalysis in the START domain superfamily, Proteins 43 (2001)134–144.

[36] N. Kudo, K. Kumagai, R. Matsubara, S. Kobayashi, K. Hanada, S. Wakatsuki, R. Kato,Crystal structures of the CERT START domain with inhibitors provide insights intothe mechanism of ceramide transfer, J. Mol. Biol. 396 (2010) 245–251.

[37] N. Kudo, K. Kumagai, N. Tomishige, T. Yamaji, S. Wakatsuki, M. Nishijima, K.Hanada, R. Kato, Structural basis for specific lipid recognition by CERT responsiblefor nonvesicular trafficking of ceramide, Proc. Natl. Acad. Sci. U. S. A. 105 (2008)488–493.

[38] P. Lavigne, R. Najmanivich, J.G. Lehoux, Mammalian StAR-related lipid transfer(START) domains with specificity for cholesterol: structural conservation andmechanism of reversible binding, Subcell. Biochem. 51 (2010) 425–437.

[39] A.P. Mathieu, P. Lavigne, J.G. LeHoux, Molecular modeling and structure-basedthermodynamic analysis of the StAR protein, Endocr. Res. 28 (2002) 419–423.

[40] S.L. Roderick, W.W. Chan, D.S. Agate, L.R. Olsen, M.W. Vetting, K.R. Rajashankar,D.E. Cohen, Structure of human phosphatidylcholine transfer protein in complexwith its ligand, Nat. Struct. Biol. 9 (2002) 507–511.

[41] M.J. Romanowski, R.E. Soccio, J.L. Breslow, S.K. Burley, Crystal structure of the Musmusculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-relatedlipid transfer domain, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 6949–6954.

[42] A.G. Thorsell, W.H. Lee, C. Persson, M.I. Siponen, M. Nilsson, R.D. Busam, T.Kotenyova, H. Schuler, L. Lehtio, Comparative structural analysis of lipid bindingSTART domains, PLoS One 6 (2011) e19521.

[43] Y. Tsujishita, J.H. Hurley, Structure and lipid transport mechanism of a StAR-related domain, Nat. Struct. Biol. 7 (2000) 408–414.

[44] D. Rodriguez-Agudo, M. Calderon-Dominguez, M.A. Medina, S. Ren, G. Gil, W.M.Pandak, ER stress increases StarD5 expression by stabilizing its mRNA and leadsto relocalization of its protein from the nucleus to the membranes, J. Lipid Res.53 (2012) 2708–2715.

U

712


ED P

RO

OF

[45] B.J. Clark, The mammalian START domain protein family in lipid transport inhealth and disease, J. Endocrinol. 212 (2012) 257–275.

[46] D. Letourneau, A. Lorin, A. Lefebvre, V. Frappier, F. Gaudreault, R. Najmanovich, P.Lavigne, J.G. Lehoux, StAR-related lipid transfer domain protein 5 binds primarybile acids, J. Lipid Res. 53 (2012) 2677–2689.

[47] A. Roostaee, E. Barbar, J.G. Lehoux, P. Lavigne, Cholesterol binding is a prerequi-site for the activity of the steroidogenic acute regulatory protein (StAR),Biochem. J. 412 (2008) 553–562.

[48] F. Delaglio, S. Grzesiek, G.W. Vuister, G. Zhu, J. Pfeifer, A. Bax, NMRPipe: a multi-dimensional spectral processing system based on UNIX pipes, J. Biomol. NMR 6(1995) 277–293.

[49] W.F. Vranken, W. Boucher, T.J. Stevens, R.H. Fogh, A. Pajon, M. Llinas, E.L. Ulrich,J.L. Markley, J. Ionides, E.D. Laue, The CCPN data model for NMR spectroscopy: de-velopment of a software pipeline, Proteins 59 (2005) 687–696.

[50] D.S. Wishart, C.G. Bigam, J. Yao, F. Abildgaard, H.J. Dyson, E. Oldfield, J.L. Markley,B.D. Sykes, 1H, 13C and 15N chemical shift referencing in biomolecular NMR, J.Biomol. NMR 6 (1995) 135–140.

[51] K.H. Gardner, M.K. Rosen, L.E. Kay, Global folds of highly deuterated, methyl-protonatedproteins bymultidimensional NMR, Biochemistry 36 (1997) 1389–1401.

[52] A. Lorin, D. Letourneau, A. Lefebvre, J.G. Lehoux, P. Lavigne, (1)H, (13)C, and (15)N backbone chemical shift assignments of StAR-related lipid transfer domain pro-tein 5 (STARD5), Biomol. NMR (2012), (Assign).

[53] H.J.C. Berendsen, D. van der Spoel, R. van Drunen, GROMACS: a message-passingparallel molecular dynamics implementation, Comput. Phys. Commun. 91 (1995)43–56.

[54] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J. Berendsen,GROMACS: fast, flexible, and free, J. Comput. Chem. 26 (2005) 1701–1718.

[55] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: algorithms for high-ly efficient, load-balanced, and scalable molecular simulation, J. Chem. TheoryComput. 4 (2008) 435–447.

[56] D. Van Der Spoel, B. Hess, GROMACS—the road ahead, WIREs Comput. Mol. Sci. 1(2011) 710–715.

[57] C.D. Berweger, W.F. van Gunsteren, F. Müller-Plathe, Force field parametrizationby weak coupling. Re-engineering SPC water, Chem. Phys. Lett. 232 (1995)429–436.

[58] C. Oostenbrink, A. Villa, A.E. Mark, W.F. van Gunsteren, A biomolecular force fieldbased on the free enthalpy of hydration and solvation: the GROMOS force-fieldparameter sets 53A5 and 53A6, J. Comput. Chem. 25 (2004) 1656–1676.

[59] C. Oostenbrink, T.A. Soares, N.F. van der Vegt, W.F. van Gunsteren, Validation ofthe 53A6 GROMOS force field, Eur. Biophys. J. 34 (2005) 273–284.

[60] A.K. Malde, L. Zuo, M. Breeze, M. Stroet, D. Poger, P.C. Nair, C. Oostenbrink, A.E.Mark, An automated force field topology builder (ATB) and repository: version1.0, J. Chem. Theory Comput. 7 (2011) 4026–4037.

[61] G. Bussi, D. Donadio, M. Parrinello, Canonical sampling through velocity rescaling,J. Chem. Phys. 126 (2007) 014101–014107.

[62] D.S. Wishart, B.D. Sykes, Chemical shifts as a tool for structure determination,Methods Enzymol. 239 (1994) 363–392.

[63] C.J. Layton, H.W. Hellinga, Thermodynamic analysis of ligand-induced changes inprotein thermal unfolding applied to high-throughput determination of ligandaffinities with extrinsic fluorescent dyes, Biochemistry 49 (2010) 10831–10841.

[64] A.F. Hofmann, The continuing importance of bile acids in liver and intestinal dis-ease, Arch. Intern. Med. 159 (1999) 2647–2658.

[65] H. Sato, A. Macchiarulo, C. Thomas, A. Gioiello, M. Une, A.F. Hofmann, R. Saladin, K.Schoonjans, R. Pellicciari, J. Auwerx, Novel potent and selective bile acid deriva-tives as TGR5 agonists: biological screening, structure–activity relationships,and molecular modeling studies, J. Med. Chem. 51 (2008) 1831–1841.

[66] G.P. Tochtrop, J.L. Bruns, C. Tang, D.F. Covey, D.P. Cistola, Steroid ring hydroxylationpatterns govern cooperativity in human bile acid binding protein, Biochemistry 42(2003) 11561–11567.

[67] O. Toke, J.D. Monsey, G.T. DeKoster, G.P. Tochtrop, C. Tang, D.P. Cistola, Determi-nants of cooperativity and site selectivity in human ileal bile acid binding protein,Biochemistry 45 (2006) 727–737.

[68] G.P. Tochtrop, G.T. DeKoster, D.F. Covey, D.P. Cistola, A single hydroxyl group gov-erns ligand site selectivity in human ileal bile acid binding protein, J. Am. Chem.Soc. 126 (2004) 11024–11029.

[69] V. Kolhatkar, J.E. Polli, Structural requirements of bile acid transporters: C-3 and C-7modifications of steroidal hydroxyl groups, Eur. J. Pharm. Sci. 46 (2012) 86–99.
N


Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated...

Documents

Transcript of Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated...