Conformational stability of 17b-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus Natas ˇ a Poklar Ulrih 1 and Tea Lanis ˇ nik Riz ˇ ner 2 1 Department of Food Science and Technology, University of Ljubljana, Slovenia 2 Institute of Biochemistry, University of Ljubljana, Slovenia Proteins of the short-chain dehydrogenase ⁄ reductase (SDR) superfamily are nonmetallo enzymes with molecular masses between 25 and 35 kDa that function as dimers or tetramers [1]. Although the SDR super- family contains some 3000 members [2], 17b-hydroxy- steroid dehydrogenase from the fungus Cochliobolus lunatus (17b-HSDcl) is currently the only fungal hydroxysteroid dehydrogenase member that has been described; it has been purified, cloned and expressed in Escherichia coli [3,4]. Under native conditions, both recombinant [4] and natural [3] 17b-HSDcl form dimers. 17b-HSDcl is homologous to some fungal reductases: versicolorin reductases from Aspergillus parasiticus and Emericella nidulans, which are involved in aflatoxin biosynthesis; and 1,3,8-trihydroxynaphthalene reductases and 1,3,6,8- tetrahydroxynaphthalene reductases from Magnaporthe grisea, Ophiostoma floccosum and other fungi, which are involved in melanin biosynthesis [4,5]. 1,3,8-Tri- hydroxynaphthalene reductases and 1,3,6,8-tetra- hydroxynaphthalene reductases catalyse an essential reaction in the biosynthesis of melanin, a virulence fac- tor of phyto-pathogenic fungi and of fungi pathogenic to humans [6–9]. These enzymes are the biochemical targets of several commercially important fungicides that are used to prevent blast disease in rice plants [9,10]. Despite extensive biochemical studies of 17b-HSDcl [4,11–14], nothing is known about its conformational stability. Structural and thermodynamic study of fun- gal 17b-HSD may, therefore, contribute to a better understanding of the functionality of homologous fun- gal enzymes that are targets for the design of novel antifungal agents. Indeed, 17b-HSDcl is often used as Keywords 17b-hydroxysteroid dehydrogenase; coenzyme NADPH binding; guanidine hydrochloride; pH stability; urea Correspondence N. Poklar Ulrih, Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Fax: +386 1 256 6298 Tel. +386 1 423 1161 E-mail: natasa.poklar@bf.uni-lj.si (Received 17 May 2006, revised 20 June 2006, accepted 26 June 2006) doi:10.1111/j.1742-4658.2006.05396.x The functional activities of proteins are closely related to their molecular structure and understanding their structure–function relationships remains one of the intriguing problems of molecular biology. We investigated struc- tural changes in 17b-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17b-HSDcl) induced by pH, temperature, salt, urea, guanidine hydrochloride, and coenzyme NADPH binding. At 25 °C and within the relatively narrow pH range of 7.0–9.0, 17b-HSDcl exists in its native conformation as a dimer. This native conformation is thermally sta- ble up to 40 °C in this pH range. At 25 °C and pH 2.0 in the presence of 150–300 mm NaCl, 17b-HSDcl forms soluble aggregates enriched in a-heli- cal and b-sheet structures. At higher temperatures and NaCl concentra- tions, these soluble aggregates start to precipitate. The denaturants urea and guanidine hydrochloride unfold 17b-HSDcl at concentrations of 1.2 and 0.4 m, respectively. Binding of the coenzyme NADPH to 17b-HSDcl causes local structural changes that do not significantly affect the thermal stability of this protein. Abbreviations C d , concentration (of urea or GuHCl) at denaturation midpoint; DG° d , Gibbs free energy of denaturation; GuHCl, guanidine hydrochloride; 17b-HSDcl, 17b-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus; DH° vH , van’t Hoff enthalpy of denaturation; SDR, short-chain dehydrogenase ⁄ reductase; T d , temperature at denaturation midpoint. FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS 3927 a model enzyme for the SDR superfamily, and fungal 17b-HSDcl exhibits  30% amino acid identity to human 17b-HSD types 4 and 8 [12]. Typically, SDR members share 15–30% residue identity in pairwise comparison. Despite the low residue identities between the different members, 3D structures revealed that the folding pattern is conserved with largely superimposa- ble peptide backbones [2]. The high similarities in these structures thus indicate that studies on 17 b-HSDcl might lead to a better understanding of the catalytic mechanisms of human HSDs that are implicated in the development of steroid-dependent forms of cancer, in polycystic kidney disease, in the regulation of blood pressure, in Alzheimer’s disease and in obesity [15–19]. This study provides the first description of the dena- turation behaviour of dimeric 17b-HSDcl, in relation to pH, temperature, ionic strength (NaCl), denaturants [urea, guanidine hydrochloride (GuHCl)] and coen- zyme binding (NADPH). Results Far-UV CD spectra of 17b-HSDcl at 25 °C Representative far-UV CD spectra of 17b-HSDcl in the pH range 1–14 are shown in Fig. 1A–C. The spec- trum of 17b-HSDcl at neutral pH (7.0; Fig. 1A) is characterized by negative CD bands near 208 and 222 nm and a positive band at 193 nm, which is typ- ical of an a-helical structure [20]. Based on contin analysis [21], at 25 °C in an aqueous solution at pH 7.0, 17b-HSDcl contains 33 ± 1% a helix (a H ), 52±1% b sheet (b S ), 15 ± 1% b turn (b T ), and 0% aperiodic secondary structure (Table 1). These values are in reasonable agreement with those predicted and obtained from molecular modelling [11] and those obtained from X-ray diffraction analysis [22]. Increas- ing the pH from neutral to an alkaline pH of 13.0, there was an alteration in the shape and intensity of Fig. 1. Far- and near-UV CD spectra of 17b-HSDcl at different pH values. (A) Far-UV CD spectra of 17b-HSDcl in the pH range 13.2–6.5. (B) Far-UV CD spectra of 17b-HSDcl in the pH range 6.5–2.8. (C) Far-UV CD spectra of 17b-HSDcl in the pH range 2.8–1.1. (D) Near-UV CD spectra of 17b-HSDcl in the pH range 7.0–2.3. Conformational stability of 17b-HSDcl N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner 3928 FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS the far-UV CD spectrum that is typical of a base- induced denaturation. At pH 13.2, 17b-HSDcl contains 6±1%a H ,34±3%b S ,11±1%b T and 49 ± 4% aperiodic structure. Only one isodichroic point was seen in the alkaline pH range, at 206.5 nm, implying the presence of two different spectroscopic states of 17b-HSDcl despite numerous deprotonation ⁄ protona- tion equilibria of the basic amino acid side chains in the alkaline pH range (Fig. 1A). Lowering the pH from neutral to an acidic pH of 1.1, there was a decrease in ellipticity, and two isodichroic points were seen at 207.5 and 206 nm (Fig. 1B,C), suggesting the existence of three spectroscopically different states of the protein. In the pH range from neutral to 2.0, there was a transition into an acid-denatured state, and a further decrease in pH from 2.0 to 1.0 induced an increase in the amount of a H structure. At pH 1.1, 17b-HSDcl contains 12 ± 2% a H , whereas at pH 2.0, it contains only 5 ± 1% a H (Table 1). Near-UV CD spectra of 17b-HSDcl at 25 °C The near-UV CD spectra of 17b-HSDcl did not change significantly in the pH range 6.0–9.0, and they are dominated by tryptophans and tyrosines (Fig. 1D). This was similar to that seen for the far-UV CD spec- tra, suggesting a stable tertiary structure of 17b-HSDcl in this pH range. pH titration of 17b-HSDcl at 25 °C The titration curves derived from signals recorded at a single wavelength at the indicated pH values are shown in Fig. 2A. The changes in molar ellipticity followed at 222 nm over the pH range 7.0–10.0 are not significant, and no changes were seen in the absorbance at 262 nm in the pH range 7.0–9.0 (Fig. 2A). In the pH range Table 1. The levels of specific secondary structure elements in 17b-HSDcl under different experimental conditions. Condition a H (%) b S (%) b T (%) AP (%) pH (at 25 °C) 13.0 6 ± 1 34 ± 3 1 ± 2 59 ± 4 7.0 33 ± 1 52 ± 1 15 ± 1 0 6.5 37 ± 2 51 ± 1 12 ± 1 0 2.0 5 ± 1 20 ± 1 9 ± 1 66 ± 1 1.1 12 ± 2 19 ± 1 9 ± 3 60 ± 3 pH 2.0 300 m M NaCl 25 °C 14±1 52±1 12±1 22±2 90 °C 0 ± 1 36 ± 4 10 ± 1 54 ± 2 pH 7.3 (at 25 °C) NADH (R ¼ 1) 33 ± 1 46 ± 2 15 ± 2 6 ± 1 A B C Fig. 2. Effects of pH on structural changes, electrophoretical prop- erties and enzymatic activity of 17b-HSDcl at 25 °C. (A) pH effects on molar ellipticity, followed at 222 nm (d), and absorbance, fol- lowed at 262 nm (s)of17b-HSDcl. (B) Electrophoretic titration ana- lysis of 17b-HSDcl in the pH range 3–9. (C) pH effect on enzymatic activity of 17b-HSDcl. N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner Conformational stability of 17b-HSDcl FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS 3929 7.0–10.0, 17b-HSDcl has a defined tertiary structure, as concluded from the near-UV CD spectra. A further increase in pH from 10.0 to 12.0 caused a sharp change in molar ellipticity followed at 222 nm, and in absorbance at 262 nm, as a consequence of disruption of the secondary and tertiary structures. We suggest that deprotonation of six Tyr (pK a ¼ 10.1), sixteen Lys (pK a ¼ 10.5) and nine Arg (pK a ¼ 12.5) residues causes loss of most of the secondary structure and a complete loss of tertiary structure at pH values above 12.0. The pK a values given in parentheses refer to the free amino acids in aqueous solution. Acid titration would be expected to result in proto- nation of 12 His (pK a ¼ 6.0), 13 Glu (pK a ¼ 4.3) and 14 Asp (pK a ¼ 3.7) residues present in 17b-HSDcl. A decrease in pH from 6.0 to 3.0 was accompanied by a reduction in molar ellipticity followed at 222 nm, which was seen as a single very broad transition, whereas two transitions were seen in the absorbance at 262 nm (Fig. 2A). Because of the 12 His, 13 Glu and 14 Asp residues in the primary structure of 17b-HSDcl, it is expected that the conformational sta- bility of 17b-HSDcl will be strongly affected by ther- modynamic coupling to the acid ⁄ base equilibrium of the acidic amino acid residues. Electrophoretic titration analysis The results of electrophoretic titration analysis of 17b-HSDcl in the pH range 3.0–9.0 are shown in Fig. 2B. Inspection of Fig. 2B reveals two transitions, the first in the pH range pH 7–5, and the second in the pH range 5–3. The observed transitions in the aci- dic pH range are likely to be the result of decreasing the net negative charge on the protein surface owing to the protonation of 12 His, 13 Glu and 14 Asp resi- dues. Inspection of Fig. 2B reveals that 17b-HSDcl in the pH range 7–3.5 is moving through the gel in two forms, most likely as a dimer and a monomer. In the pH range 7.0–9.0 (pI ¼ 6.9) the net charge on the pro- tein remains constant and 17b-HSDcl does not move in the electric field. Enzymatic activity The results of enzymatic activity of recombinant 17b-HSDcl in the pH range 6.0–8.5 followed by oxida- tion of 4-estrene-17b-ol-3 one to 4-estrene-3,17-dione in the presence of NADP + are shown in Fig. 2C. The pH optimum of 17b-HSDcl is between 7 and 8, as shown previously for the enzyme, which was isolated directly from the fungus Cochliobolus lunatus [3]. Acid-induced denatured state of 17b-HSDcl at 25 °C As seen in Fig. 2A, in the pH range 2.0–3.5, the molar ellipticity at 222 nm and absorbance at 262 nm of 17b-HSDcl did not change significantly, suggesting that 17b-HSDcl was in a stable conformation, acid- unfolded state, designated U A . At pH values < 2.0, molar ellipticity gradually increased (became more negative), and at pH 1.1, 17b-HSDcl retained a more ordered secondary structure than at pH 2.0 (Table 1). The results from native PAGE electrophoresis of 17b- HSDcl incubated in 1 m HCl indicate that the increase in secondary structure at pH values below 2.0 is likely to be due to the oligomerization processes (Fig. 4A,B, column 5). Salt-induced effects on 17b-HSDcl at pH 2.0 The addition of a neutral electrolyte, such as NaCl, could shield repulsion interactions in the highly posi- tively charged 17b-HSDcl at pH 2.0. The effects on the secondary structure of 17b-HSDcl of increasing NaCl concentrations at pH 2.0 and 25 °C are shown in Fig. 3A, and these indicate that an increase in NaCl concentration induces an increase in b S structure. At 300 mm NaCl, the a H and b S structures increase from 5 to 14% and 20 to 52%, respectively (Table 1). The inset in Fig. 3A shows the changes in molar ellipticity followed at 215 nm vs. NaCl concentration. Clearly the transition from the U A state (stable conformation, acid unfolded) to the conformational state with a non- native secondary structure, occurs in the NaCl concen- tration range 150–300 mm. The thermal stability of this newly formed soluble oligomers of 17b-HSDcl at pH 2.0 in the presence of 300 mm NaCl (Fig. 4A,B, column 4) was investigated by measuring the CD spec- tra at different temperatures in the far-UV range (Fig. 3B). The obtained results show that oligomers of 17b-HSDcl are resistant to temperatures up to 60 °C. At temperatures above 60 °C, gradual changes in molar ellipticity were seen (Fig. 3B), suggesting the formation of larger aggregates or disruption of 17b-HSDcl oligomers containing significant amounts of non-native secondary structure. The results from native and SDS ⁄ PAGE confirm that 17b-HSDcl at pH 2.0 does not oligomerize in the absence of salt, whereas in the presence of 100 and 300 mm NaCl 17b-HSDcl is in oligomeric form (Fig. 4A,B). The oligomers of 17b-HSDcl in the pres- ence of NaCl are too large to penetrate into the gel (Fig. 4A), whereas under the denaturing conditions Conformational stability of 17b-HSDcl N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner 3930 FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS they dissociate into monomeric form (Fig. 4B). At NaCl concentrations > 350 mm, the precipitation of 17b-HSDcl has been observed in the cuvette. Heat-induced denaturation of 17b-HSDcl at pH 7.0–8.4 The pH titration of 17b-HSDcl at 25 °C indicated a very narrow pH range at which the 17b-HSDcl tertiary and secondary structures are intact (see above). In this narrow pH range from 7 to 8.5, we studied thermal stability of 17b-HSDcl from CD and UV melting curves, from which the temperature of denaturation (T d ) and the van’t Hoff enthalpy of denaturation (DH° vH )of17b-HSDcl were obtained, as described previously [23]. DH° vH was calculated based on assumption that the thermal denaturation of 17b- HSDcl is a reversible two-state process. In fact, heat- induced denaturation of 17b-HSDcl was reversible if the experiment was stopped immediately after the transition temperature. The degree of reversibility decreased with the temperature to which the sample of 17b-HSDcl was heated (data not shown). The thermodynamic profile of 17b-HSDcl is given in Table 2. In the pH range 7.0–8.0, the T d and DH° vH of 17b-HSDcl do not change significantly. From the UV melting profile, the T d for 17b-HSDcl at pH 7.5 is rel- atively low, at 42.9 ± 0.5 °C; this is perhaps not so surprising as it has been shown that the apparent opti- mal temperature of enzymatic activity of 17b-HSDcl is 28 °C at pH 7.0 [3]. Slightly higher T d and DH° vH values were determined from the CD melting profiles (Table 2). Urea and GuHCl effects on 17b-HSDcl at 25 °C The effects of increasing concentrations of urea and GuHCl on the structural properties of 17b-HSDcl were investigated using far- and near-UV CD. The results presented in Fig. 5A–D indicate that 17b-HSDcl unfolds at very low concentrations of denaturants. The denaturation concentrations at which half of the 17b- HSDcl molecules are in the denaturated and a half in the native state, C d , are 1.2 m for urea and 0.4 m for Gu- HCl (determined from Fig. 5A,B). Because the denatu- rant-induced unfolding of 17b-HSDcl was a reversible two-state transition, it can be described in terms of its equilibrium constant, K d °. From these K d ° values, the corresponding Gibbs free energies, DG d °,of17b-HSDcl in solutions of different concentrations of urea and GuHCl can be determined using the general relation: DG d ° ¼ –RT lnK d °. Numerous studies on urea and GuHCl denaturation of proteins have shown that in the denaturant concentration range in which the denatura- tion of proteins can be followed, the DG d ° of denatura- tion can be expressed as a linear function of denaturant concentration: DG d  ¼ DG d ;H 2 O  mC [24,25]. For 17b-HSDcl, the calculated m-values at 25 °C and pH 7.0 are )12.9 ± 0.6 kJÆLÆmol )1 for urea and )14.4 ± 0.8 kJÆLÆmol )1 for GuHCl, whereas corresponding val- ues for are 15.3 ± 0.7 and 5.9 ± 0.3 kJÆmol )1 , respect- ively. These DG d ;H 2 O values obtained from analysing the data for urea and GuHCl denaturation of 17b- HSDcl give DG d ;H 2 O from GuHCl as threefold lower than that from urea. Possible explanations for this pheno- menon could arise from a lack of the native baseline and the errors involved in the d ata c ollection and analysis. AB Fig. 3. Effects of NaCl on structural changes in 17b-HSDcl at pH 2.0. (A) NaCl concentration effects on far-UV CD spectra and molar ellipticity followed at 215 nm (inset)of17b-HSDcl at pH 2.0. (B) Temperature effects on molar ellipticity followed at 235 nm ( ) and 217 nm (s)of 17b-HSDcl in the presence of 300 m M NaCl at pH 2.0 (in 10 mM glycine buffer). N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner Conformational stability of 17b-HSDcl FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS 3931 NADPH binding to 17b-HSDcl at 25 °C The effects of the coenzyme NADPH on the confor- mation of 17b-HSDcl were investigated at pH 7.3 in NaCl ⁄ P i buffer. The influence of NADPH on the far- UV CD spectrum of 17b-HSDcl at a molar ratio of 1 : 1 for NADPH ⁄ 17b-HSDcl (per monomeric unit) can be seen in Fig. 6A. The changes in the secondary structure of 17b-HSDcl after NADPH binding are not significant (Fig. 6B), suggesting that the coenzyme interrupts the structure of 17b-HSDcl only locally. Binding of the coenzyme NADPH to 17b-HSDcl at a molar ratio of 1 : 1 thermally stabilized 17b-HSDcl for 0.6 ± 0.5 °C, whereas at higher NADH:17b- HSDcl molar ratios, e.g. 5 : 1, the thermal stabiliza- tion was 2.5 ± 0.5 °C. These data are in agreement with the CD data, which indicate only minor struc- tural rearrangements of 17b-HSDcl after NADPH binding. Discussion For oligomeric proteins changes in secondary and ter- tiary structures during native to denatured transitions are usually accompanied by dissociation into subunits. 17b-HSDcl is a dimeric member of the SDR super- family, in which neighbouring subunits are connected via hydrophobic interactions and several salt bridges involving amino acid residues His111 and Arg129. It has been shown that Arg129 and His111 interact with Asp121, Glu117 and Asp187 residues from the neigh- bouring subunits [14]. Replacement of His111 with Leu prevented dimer formation and caused loss of bio- logical activity of 17b-HSDcl, whereas the His111Ala mutation did not affect either dimerization or enzyme activity. It has also been reported [3] and confirmed by our measurements that 17b-HSDcl is active in the pH range 7.0–9.0. The results reported here show that the conformational changes coincide with the changes in functional activity. A loss in enzymatic activity for 17b-HSDcl at pH values < 7.0, which is at first seen as a slight change in the far-UV CD signal in the pH range 7.0–6.0 and it is followed by significant change in the CD and UV signal, can be ascribed to denatura- tion of 17b-HSDcl. The results from electrophoretic titration analysis show that 17b-HSDcl in the pH range 7–3.5 moves through the gel in two forms. Although the dimeric form of 17b-HSDcl is predomi- nant, we proposed that partial dissociation is taking place at pH values < 7.0 and it is likely to be induced by protonation of the His111 residue that is involved in dimerization. More precisely, it has been proposed that dimerization takes place across the Q-axis and involves the a-E and a-F helices of both subunits [14]. Our CD data show a slight increase in the amount of a H structure in the pH range 7.0–6.5 and further sup- port the results from electrophoretic titration analysis that 17b-HSDcl partially dissociate into subunits at pH values < 7.0. Table 2. The thermodynamic profile of 17b-HSDcl obtained from UV melting curves at different pH values in NaCl/P i buffer. a from CD measurements. T d , temperature at denaturation midpoint; DT, the width of the transition; DH° vH , van’t Hoff enthalpy of denaturation. pH T d (°C) DT(°C) DH° vH (kJ ⁄ mol) 7.3 a 47.0 ± 0.5 7.2 ± 1 355 ± 30 7.0 41.8 ± 0.5 11.4 ± 1 289 ± 30 7.5 42.9 ± 0.5 11.7 ± 1 285 ± 30 8.0 42.8 ± 0.5 11.6 ± 1 288 ± 30 8.4 44.9 ± 0.5 12.0 ± 1 279 ± 30 BSA A 12 3 4 5 st.B 12 3 4 5 Fig. 4. Native and SDS ⁄ PAGE electrophoretogram of 17b-HSDcl. A total of 8 lg of recombinant 17b-HSDcl in the following solutions: (1) NaCl ⁄ P i buffer, pH 7.3; (2) 10 mM glycine buffer, pH 2.0; (3) 10 m M glycine buffer, 100 mM NaCl, pH 2.0; (4) 10 mM glycine buf- fer, 300 m M NaCl, pH 2.0 and (5) 1 M HCl were applied to (A) native and (B) SDS ⁄ PAGE. BSA was used for comparison on native PAGE. Prestained molecular markers were: 20, 26, 36, 47, 85 and 118 kDa, respectively. Conformational stability of 17b-HSDcl N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner 3932 FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS High aggregation ability is a conventional property of non-native protein conformations. Examples include aggregation following heat- or pH-induced denatura- tion and aggregation of folding intermediates, and this has been observed for numerous globular proteins. Under conditions of extreme pH, the main forces that unfold proteins are repulsion between charged groups in the protein molecule. The 17b-HSDcl molecule is highly charged (the absolute net charge at pH 2.0 esti- mated from its amino acid composition is +31), and therefore the acidic unfolded U A state does not aggre- gate. The observed increase in molar ellipticity at pH values < 2.0 indicates some electrostatically driven structural changes in this protein molecule in response to an increased concentration of Cl – ions [26], which is further supported by the NaCl titration of 17b-HSDcl at pH 2.0. After addition of salt, formation of b S structures is observed, as results of the oligomerization processes. The presence of high concentrations of salt has two effects on protein–protein interactions: First, the presence of counterions around the charged groups weakens the repulsion and allows intermolecular forces become relatively strengthened and thus manifest themselves [26]. In addition to this nonspecific effect of salt as counterions, specific effects of salt on protein– protein interactions indicate the presence of exposed hydrophobic surfaces [26]. Additional information relating to the presence or absence of a unique tertiary structure of a protein molecule can be obtained from analysis of its urea- and GuHCl-induced unfolding. Indeed, it has been shown that the steepness of a denaturant-induced unfolding curve depends strongly on whether a given protein has a unique tertiary structure or whether it is Fig. 5. Urea- and GuHCl-induced denaturation of 17b-HSDcl at 25 °C. (A) Urea and (B) GuHCl effects on far-UV and (C) near-UV CD spectra of 17b-HSDcl. (D) Urea (d) and GuHCl (s) effects on molar ellipticity followed at 220 nm of 17b-HSDcl. N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner Conformational stability of 17b-HSDcl FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS 3933 already denatured and exists as a molten globule [27]. Urea and GuHCl induced unfolding of 17b-HSDcl at C d values of 1.2 and 0.4 m, respectively. Similarly, a C d value of 0.64 m was reported by Oppermann et al. [28] for GuHCl denaturation of 3b ⁄ 17b-HSD from Comamonas testosteroni, with a corresponding value of 15.1 kJÆmol )1 which is comparable with our value of 15.3 kJÆmol )1 [28]. Human placenta 17b-hydroxyster- oid-dehydrogenase (17b-HSD type 1), which shares 21% amino acid identity with 17b-HSDcl and posses- ses the same protein fold, has urea-induced conforma- tional transitions with C d values of 1.5 and 5.8 m, suggesting that the first transition is from the native dimeric state to a molten-globule-like dimeric state, and that the second transition leads to the fully dena- tured state that is accompanied by the dissociation of oligomeric molecules [29]. NADPH-dependent enzymes have one or two con- served basic residues that interact electrostatically with the ribose 2¢-phosphate group of the adenine nucleo- tide of NADPH. One of these interacts with the sec- ond glycine in the Gly-X-X-X-Gly-X-Gly motif, and this is restricted to a Lys or Arg, which prevails in lower organisms [30,31]. In 17b-HSDcl, Arg28 com- pensates for the negative charge of the 2¢-phosphate group of the adenine nucleotide of NADPH, whereas Thr200 and Thr202 interact with the nicotinamide moiety of NADPH [13]. Our results show that the co- enzyme NADPH clearly binds to the free enzyme. The dissociation constant, K d , of the enzyme-NADPH complex of 1.6 lm has been previously determined by fluorescence measurements at pH 8.0 in a 100 mm phosphate buffer [13]. The changes in the secondary structure of 17b-HSDcl after binding of NADPH are not significant, although a slight increase in the aperio- dic structure seen at the expense of b S structure is seen. This local rearrangement of secondary structure does not significantly affect the thermal stability of 17b- HSDcl. Indeed, a previous report [28] that binding of NAD + to 3b ⁄ 17b-HSD from C. testosteroni is influ- enced by local structural changes, involving strand b D and turn b A to a B , supports our data. In conclusion, our study indicates that 17b-HSDcl is enzymatically active and thermodynamicly stable over a narrow pH range, as would be the case for other proteins in the SDR superfamily that function as dimers or tetramers. The loss of enzymatic activ- ity of 17b-HSDcl at pH values < 7.0 can be ascribed to protonation ⁄ deprotonation equilibria of numerous acidic amino acid residues causing the denaturation of 17b-HSDcl. The combined results of the unfolding of 17b-HSDcl suggest that it can take on different conformational states at 25 °C, as sum- marized by the scheme: Agg(i) Agg(s) $ U A $ N 2 $ D B where Agg(i) is the insoluble aggregates of 17b-HSDcl (pH £ 2.0 and concentration of NaCl > 300 m m); Agg(s), soluble oligomers of 17b-HSDcl in the pres- ence of salt (pH ¼ 2.0 and 150–300 mm NaCl); U A , the acid-unfolded state (pH 2–3); N 2 , the native dimeric state (pH 7–9); and D B , the base-denatured state at pH > 10. Of note, the observed thermody- namic stability of 17b-HSDcl at 25 °C, with a value of 15.3 kJÆmol )1 (0.06 kJÆmol )1 amino acid), is much lower than for the majority of globular proteins ( 0.4 kJÆmol )1 amino acid). The binding of the Fig. 6. Coenzyme NADPH binding to 17b-HSDcl at 25 °C. (A) Coen- zyme NADPH effects on far-UV CD spectra of 17b-HSDcl at 25 °C at a molar ratio of 1 : 1. (B) The molar ellipticity of 17b-HSDcl at 220 nm, [Q] 220 , as a function of increasing NADPH concentration expresed as molar ratio R ([NADPH] ⁄ [17b-HSDcl]) at 25 °C. Conformational stability of 17b-HSDcl N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner 3934 FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS coenzyme NADPH induces local structural reorganiza- tion of 17b-HSDcl without significantly influencing this thermal stability. These data thus show that the absence of induction of thermal stability by NADPH binding is the consequence of enthalpic compensation of the disturbed intramolecular interactions by the newly formed electrostatic and H-bonds with NADPH, as was suggested by earlier structural modelling of 17b-HSDcl based on trihydroxynaphthalene reductase from Magnaporthe grisea [11]. Experimental procedures Materials NADPH, glycine, hystidine and Mes were from Sigma-Ald- rich (St. Louis, MO). GuHCl and urea were from Fluka (Buchs, Switzerland). GuHCl was recrystallized from hot ethanol before use. Na 2 HPO 4 and NaH 2 PO 4 were from Kemika (Zagreb, Croatia), NaCl, NaOH and HCl for the titration experiments and dimethyl formamide were from Merck (Darmstadt, Germany). Solutions NADPH solutions in NaCl ⁄ P i buffer and double-distilled water were prepared immediately before use. The concen- trations of NADPH were determined spectrophotometrical- ly at 340 nm and 25 °C, using an extinction coefficient of 65 000 m )1 Æcm )1 . NaCl ⁄ P i buffer (142.7 or 300 mm NaCl, 10 mm Na 2 HPO 4 , 1.8 mm NaH 2 PO 4 , pH 7.3), 10 mm gly- cine buffer (pH 2.0), and double-distilled water were used as solvents. Recombinant 17b-HSDcl Recombinant 17b-HSDcl was prepared as a GST-fusion protein in the Escherichia coli strain JM107 and purified using affinity chromatography on glutathione–Sepharose, followed by cleavage with thrombin, as described previously [4]. The 17b-HSDcl concentration was determined spectro- photometrically at 280 nm and 25 °C using a calculated extinction coefficient [32] of 20 065 m )1 Æcm )1 (per monomer unit). Enzymatic assay Enzymatic activity of the recombinant 17b-HSDcl was determined spectrophotometrically at 340 nm. We fol- lowed the oxidation of 4-estrene-17b-ol-3-one to 4-est- rene-3,17-dione (Sigma-Aldrich) in the presence of NADP + in 100 mm phosphate buffer, pH 6–8.5 at 25 °C. In all of the experiments, 1% dimethyl formamide was present to enhance substrate solubility. The time course of absorbance was measured for 450 s and initial veloci- ties were determined. Denaturation studies Temperature-, pH-, urea- and GuHCl-induced denaturation of 17b-HSDcl were monitored using a combination of UV spectrophotometry and CD measurements. UV spectrophotometry The UV light absorbance values were measured using a Hewlett Packard 8453 UV-VIS spectrophotometer (Hewlett- Packard GmbH, Waldbronn, Germany) equipped with a thermoelectrically controlled cell holder. The UV-absorption spectra of 17b-HSDcl were measured after titration with HCl or NaOH. The absorbance vs. temperature profiles of 17b-HSDcl were measured at 280 nm. Temperature was increased in 1 °C increments, and protein samples were allowed to equilibrate for 1 min at each temperature setting. The temperature-induced denaturation profiles of 17b-HSDcl were used to determine transition temperatures, T d and van’t Hoff enthalpy of denaturation, DH v.H . The subsequent absorbance vs. temperature profiles of 17b-HSDcl were used to assess the reversibility of the protein denaturation. CD The CD spectra were measured using an AVIV Model 62 A DS spectropolarimeter (AVIV Associates, Lakewood, NJ) equipped with a thermoelectrically controlled cell holder. Cuvettes with path lengths of 1 mm were used for far- (200–260 nm) and 10 mm for near-UV (240–310 nm) measurements, with 17b-HSDcl concentrations of 0.25 and 0.75 mgÆmL )1 , respectively. CD spectra were recorded either as functions of temperature, between 10 and 95 °Cin 5 °C steps, or of pH (HCl or NaOH) or ion (NaCl), denat- urant (urea, GuHCl) and coenzyme (NADPH) concentra- tions. The latter were achieved by incremental additions of the relevant reagents to a cuvette containing a known amount of 17b-HSD at 25 °C. The mean residue ellipticity, [h] k , was calculated by using the relation: ½H k ¼ M O H k 100  c  1 ð1Þ in which M o is the mean residue molar mass (107.0 gÆmol )1 for 17b-HSDcl), Q k is the measured ellipticity in degrees, c is the concentration in gÆmL )1 , and l is the path length in decimetres. [ Q] k was expressed in deg cm 2 Ædmol )1 . Secon- dary structure content was calculated from the far-UV CD spectra using contin software [21]. The degree of reversibil- ity of the urea- and GuHCl-induced unfolding of 17b-HSDcl was determined by measuring the CD spectrum of 17b-HSDcl after dialysing the sample of protein in urea or GuHCl against buffer solution. N. Poklar Ulrih and T. Lanis ˇ nik Riz ˇ ner Conformational stability of 17b-HSDcl FEBS Journal 273 (2006) 3927–3937 ª 2006 The Authors Journal compilation ª 2006 FEBS 3935 pH measurements The pH titration was performed at 25 °C using a 10-lL Hamilton syringe (Hamilton Co., Reno, NV) equipped with a Chaney adapter. The pH of protein solutions was meas- ured separately using a pH-meter (model MA 5705; Iskra, Slovenia) with an Ag ⁄ AgCl combination microelectrode (Mettler, Toledo, Spain). The absolute error of pH meas- urements was ± 0.01 pH units. Native PAGE Samples of 17 b-HSDcl in: (a) 10 mm glycine buffer, pH 2.0 in the absence or presence of NaCl (100, 300 mm); (b) 1 m HCl; (c) in NaCl ⁄ P i buffer, pH 7.3. were analysed by native PAGE. Discontinous native PAGE was performed on 9% acrylamide gels according to Ornstein-Davis in 25 mm Tris, 190 mm glycine pH 8.3 buffer [33]. Continous native PAGE was carried out on 9% acrylamide gels in 30 mm histidine, 30 mm Mes buffer pH 6.1 [34]. Following electrophoresis at 150 V, the proteins were stained using Coomassie Brilliant Blue. SDS/PAGE Samples of 17b-HSD (see above) were analysed also using SDS ⁄ PAGE. Eight micrograms were denatured in sample buffer and then applied to 12% acrylamide gel [35]. After ectrophoresis at 200 V, the proteins were stained using Coomassie Brilliant Blue. Electrophoretic titration analysis Electrophoretic titration curve analysis is a 2D technique that allows the determination of protein charge characteris- tics. It was performed using a PhastGel IEF 3–9 media. In the first dimension, the stable linear pH gradient was generated. The gel was then rotated 90° clockwise and 17b-HSDcl was applied perpendicularly to the pH gradient across the middle of the gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and documented. 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Conformational stability of 17b-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus Natas ˇ a Poklar Ulrih 1 and. remains one of the intriguing problems of molecular biology. We investigated struc- tural changes in 17b-hydroxysteroid dehydrogenase from the fungus Cochliobolus