Báo cáo khoa học: Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B pot

Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B Young S. Yun 1,2 , Gyu H. Nam 1,2 , Yeon-Gil Kim 1,3 , Byung-Ha Oh 1,3 and Kwan Y. Choi 1,2 1 Division of Molecular and Life Sciences, Pohang University of Science and Technology, South Korea 2 National Research Laboratory of Protein Folding and Engineering, Pohang University of Science and Technology, South Korea 3 National CRI Center for Biomolecular Recognition, Pohang University of Science and Technology, South Korea Hydrophobic residues are rarely found on the surface of soluble globular proteins because their exclusion from water is favored by the hydrophobic effect [1]. However, stabilizing effects resulting from the intro- duction of hydrophobic residues on the surface of a protein have been observed [2–4]. A hydrophobic sur- face formed by hydrophobic side-chains was found to be associated with the formation and stabilization of the overall b-sheet structure [5]. A structural motif known as the small exterior hydrophobic cluster Keywords conformational stability; ketosteroid isomerase; small exterior hydrophobic cluster; steroid binding; surface hydrophobic residue Correspondence K. Y. Choi, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, South Korea Fax: +82 54 279 2199 Tel: +82 54 279 2295 E-mail: kchoi@postech.ac.kr Database The atomic coordinate and structural factor of W92A have been deposited in the Protein Data Bank under the access code 1W6Y. (Received 5 November 2004, revised 26 January 2005, accepted 24 February 2005) doi:10.1111/j.1742-4658.2005.04627.x A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent- exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in D 5 -3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side-chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI-PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A ⁄ V127A, and W92A ⁄ L125A⁄ V127A mutations largely decreased the conformational sta- bility, while the L125F ⁄ V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in main- taining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side-chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent-exposed b-sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI-PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the U U value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent-exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI-PI to stabilize the hydro- phobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution. Abbreviations 5-AND, 5-androstene-3,17-dione; K D , dissociation constant; d-equilenin, d-1,3–5(10),6,8-estrapentaen-3-ol-17-one; KSI, D 5 -3-Ketosteroid isomerase; KSI-PI, KSI from Pseudomonas putida biotype B; 19-nortestosterone, 17b-hydroxy-4-estren-3-one; SEHC, small exterior hydrophobic cluster; WT, wild type. FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS 1999 (SEHC) was suggested to explain the stabilizing effect of the hydrophobic residues at a solvent-exposed site [6]. In nonsequential b-strands, the SEHC may con- tribute to conformational stability and folding by organizing a small cluster to fix the b-strands on the protein surface. Such a hydrophobic cluster on the protein surface may be used in the rational design of proteins to increase conformational stability [7,8]. The protein surface is the significant site for inter- action of the protein with ligands and substrates [9]. Recent studies have shown that solvent-exposed hydro- phobic residues or clusters are important in protein– ligand interactions in which hydrophobic residues can interact directly with the hydrophobic moieties of lig- ands at solvent-exposed sites [10,11]. In the case of an enzyme that converts large hydrophobic substrates, molecular recognition between the hydrophobic sub- strate and the hydrophobic surface of the enzymes is required prior to the enzyme reaction. However, hydrophobic residues that are exposed to solvent for hydrophobic substrate binding may inevitably destabil- ize protein stability. Interfacial activation via an amphiphilic lid has been proposed to explain the bind- ing and activation of lipids in some lipases [12–14] and in cholesterol oxidase [15–17]. This type of recognition involves the opening of the amphiphilic lid to expose the hydrophobic surface towards the solvent, leading to the binding of lipids or cholesterols. Simultaneously, the lid opening can lead to activation of the enzymes by reorganization of the active site. Steroids are important hydrophobic molecules that play significant roles as hormones or transcription factors together with their receptor proteins. However, the mechanism that determines binding affinity or specificity is poorly understood in steroid-binding or steroid-converting proteins [18]. D 5 -3-Ketosteroid isomerase (KSI; EC 5.3.3.1) has been reported to contain hydrophobic residues at sol- vent-exposed sites [19]. KSI catalyzes a reaction from D 5 -3-ketosteroids to D 4 -3-ketosteroids at a diffusion- controlled limit (Scheme 1) [20,21]. In animals, this reaction is essential for the synthesis of steroid hormones from cholesterol. Two KSIs from different bacteria – Pseudomonas putida biotype B and Coma- monas testosteroni – have been studied extensively as prototypes in order to understand, in greater detail, the catalytic mechanism of the allylic rearrangement [21–28]. X-ray crystal structures [19,29] and the NMR solution structure [30] of KSI have revealed that this protein folds into a six-stranded b-sheet and three a-helices in each monomer (Fig. 1A). One of the most noticeable features of KSI from P. putida (KSI-PI) is that the bulky side-chain of Trp92 forms a hydropho- bic cluster with the aliphatic side-chains of Leu125 and Val127 on the surface. Leu125 and Val127 are located close to the C-terminal end, and the hydrophobic clus- ter is located at the center of three b-strands (B4, B5 and B7) that are exposed to solvent (Fig. 1B) [19]. Interestingly, this SEHC is located on top of the coni- cal cleft of the active site in KSI-PI. Even though KSI- PI exposes the hydrophobic residues to solvent, it exhibits high thermodynamic stability (a DG H 2 O U value of 24 kcalÆmol )1 ) and is highly soluble, without aggre- gation at high concentration [31,32]. In this study, the SEHC in KSI-PI was characterized to identify its roles in conformational stability and steroid binding. The SEHC was perturbed by site- directed mutagenesis in order to investigate the muta- tional effects on catalysis, stability, unfolding and binding affinity of KSI-PI. The crystal structure of W92A in complex with d-1,3–5(10),6,8-estrapentaen-3- ol-17-one (d-equilenin), determined at 2.1 A ˚ resolution, provided a structural basis for understanding the roles of the SEHC. Our studies demonstrate that the SEHC in KSI-PI is required to stabilize the surface conforma- tion of solvent-exposed b-strands, thereby contributing to the overall conformational stability and the binding affinity of steroids. O O O O O O H H H H OOH O O OO OOH OO H Tyr14 OH Tyr14 OH Tyr14 OH O OH Asp99 Asp38 Asp99 Asp38 Asp99 Asp38 4 6 Scheme 1. General catalytic mechanism of D 5 -3-ketosteroid isomerase (KSI). The residues are numbered according to Comamonas testo- steroni KSI in this scheme. Role of small exterior hydrophobic cluster in KSI Y. S. Yun et al. 2000 FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS Results Structure of the SEHC Based on the crystal structure of KSI-PI, the residues Trp92, Leu125, and Val127 were found to be exposed to solvent. Using a probe radius of 1.4 A ˚ , the acces- sible surface areas of the side-chains of Trp92, Leu125, and Val127 were calculated to be 203.4, 85.7, and 75.8 A ˚ 2 , respectively. Given that the maximum access- ible surface areas for a maximally exposed side-chain were determined to be 282.5, 197.5, and 179.3 A ˚ 2 , the side-chains of Trp92, Leu125, and Val127 are 72.0, 43.4, and 42.3% exposed, respectively. Moreover, an SEHC consisting of Trp92, Leu125, and Val127 was found to be located on the center of three b-strands (B4, B5 and B7) that occupy the entry site of the ster- oid-binding pocket (Fig. 1B). Mutational effect on catalysis To investigate the mutational effects of W92A, L125A ⁄ V127A, W92A ⁄ L125A ⁄ V127A, and L125F ⁄ V127F on the catalytic parameters, the k cat and K M values of the mutant KSI-PIs were determined using 5-androstene-3,17-dione (5-AND) as a substrate (Table 1). The removal or addition of hydrophobic moieties from the SEHC affected the K M more than the k cat . The k cat values decreased by 1.6-, 1.6-, 1.7- and 1.2-fold for W92A, L125A ⁄ V127A, W92A ⁄ L125A ⁄ V127A and L125F ⁄ V127F while the K M values increased by 1.9-, 2.7-, 2.3- and 1.5-fold, respectively, indicating that the SEHC could affect the substrate- binding step as well as the catalytic step in the enzyme reaction. Effect of mutations on conformational stability The unfolding free-energy change, DG U , was deter- mined by monitoring the molar ellipticity of the pro- teins at 222 nm upon changing the urea concentration at 25 °C. The transition curves were normalized by assuming that ellipticities for the native and unfolded state can be extrapolated linearly into the transition zone and nicely fitted to a two-state transition model (Fig. 2). By applying the two-state transition model, the values of DG H 2 O U , m, and DDG U for wild-type (WT) and mutant enzymes were obtained (Table 2). Removal of hydrophobic moieties from the SEHC decreased the DG H 2 O U values by 3.1 and 4.2 kcalÆmol )1 Table 1. Kinetic parameters of the wild-type (WT) enzyme and its mutants for the isomerization of 5-androstene-3,17-dione to 4-androstene- 3,17-dione. The assays were performed in buffer containing 34 m M potassium phosphate and 2.5 mM EDTA, pH 7.0. Enzyme k cat (s )1 ) K M (lM) k cat ⁄ k M (M )1 Æs )1 ) Relative k cat a Relative k M b WT (21.2 ± 0.8) · 10 3c 49.9 ± 1.3 c 4.3 · 10 8 1.000 1.000 W92A (12.7 ± 1.9) · 10 3 99.5 ± 23.7 1.3 · 10 8 0.599 1.993 L125A ⁄ V127A (12.9 ± 0.5) · 10 3 137.5 ± 10.3 0.9 · 10 8 0.608 2.755 W92A ⁄ L125A ⁄ V127A (12.0 ± 0.8) · 10 3 118.5 ± 8.7 1.0 · 10 8 0.566 2.374 L125F ⁄ V127F (17.6 ± 0.8) · 10 3 79.5 ± 3.8 2.3 · 10 8 0.827 1.593 a,b Values relative to those of the WT enzyme. c Data from [42]. Val127 Val127 Leu125 Leu125 Trp92 Trp92 d-equilenin d-equilenin A B Fig. 1. Structure of D 5 -3-ketosteroid isomerase from Pseudomon- as putida biotype B (KSI-PI). (A) Ribbon diagram of the dimeric structure of KSI-PI in complex with d-equilenin. (B) Stereoview of the monomeric structure of the dimeric KSI-PI. Trp92, Leu125, Val127, and d-equilenin are displayed by a ball-and-stick model. The figures were drawn using the program SWISS-PDB VIEWER, Version 3.7 [49]. Y. S. Yun et al. Role of small exterior hydrophobic cluster in KSI FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS 2001 in W92A and W92A⁄ L125A ⁄ V127A, respectively. However, the L125F ⁄ V127F mutation of the SEHC increased the DG H 2 O U value by 0.9 kcalÆmol )1 , while the L125A ⁄ V127A mutation of the SEHC decreased the DG H 2 O U value by 3.3 kcalÆmol )1 . These results indi- cate that the SEHC formed by Trp92, Leu125 and Val127 contributes to the conformational stability. Unfolding kinetics The unfolding of the enzymes was monitored by meas- uring the fluorescence intensity, as a function of time, at various urea concentrations. The unfolding curve was nicely fitted to Eqn (6). When plots of ln k U vs. the urea concentration were made in the range where the proteins are more than 95% unfolded at equilib- rium, straight lines were obtained (Fig. 3). The unfold- ing rates of W92A, L125A ⁄ V127A and W92A ⁄ L125A ⁄ V127A were faster than that of the WT enzyme. How- ever, the unfolding rate of L125F ⁄ V127F was slower than that of the WT enzyme, suggesting that the hydro- phobic moieties may play a role during the unfolding process. The free-energy change of the unfolding trans- ition state was assessed from the unfolding rate con- stants (Table 3). Analyses of the transition-state interaction The hydrophobic interaction of the SEHC during the unfolding process was investigated by U U value analy- sis, according to the method described previously [33]. The U U value can range from 0 to 1. A high U U value implies that the target region is exposed to solvent in the transition state to the same extent as in the unfol- ded state, while a low U U value implies that the inter- action energies in the transition states and folded states are similar. The W92A, L125A ⁄ V127A and W92A ⁄ L125A ⁄ V127A mutants gave U U values of 0.451, 0.393 and 0.500, respectively, indicating that 50.0–60.7% of the noncovalent interaction energy is maintained in the transition state for the hydrophobic cluster (Table 3). However, the L125F ⁄ V127F mutant had a relatively high U U value of 0.777. The ratio of m U à ⁄ m U has been reported to indicate the increase in solvent exposure of the transition state relative to the native state [34]. The m U à ⁄ m U values of the WT enzyme were determined to be 0.147, indicating that the solvent accessibility of the transition state is very similar to that of the native state. Effect of mutations on d-equilenin binding d-Equilenin has a maximum emission peak at 363 nm when excited at 335 nm. Addition of the enzyme Fig. 2. Unfolding equilibrium transition of the wild-type (WT) enzyme (s), and those of the mutants W92A (n), L125A ⁄ V127A (·), W92A ⁄ L125A ⁄ V127A (e), and L125F ⁄ V127F (h). The fraction of unfolded protein at each urea concentration was calculated from the molar ellipticity at 222 nm after correction for the pre- and post- transition baselines. The transition curves were obtained by fitting the data to Eqn (4). Table 2. Changes in the free energies of unfolding of the wild-type (WT) enzyme and its mutants in the reversible denaturation with urea. Measurements were performed at 25 °C and pH 7.0. Values were obtained by fitting the data from Fig. 2 according to Eqn (4). Enzyme DG U H 2 Oa (kcalÆmol )1 ) m b (kcalÆmol )1 ÆM) [Urea] 50% c (M) DG U d (kcalÆmol )1 ) WT 24.0 ± 0.5 3.41 ± 0.06 5.22 ± 0.12 W92A 20.9 ± 0.4 3.38 ± 0.05 4.20 ± 0.10 ) 3.1 L125A ⁄ V127A 20.7 ± 0.3 3.26 ± 0.04 4.21 ± 0.07 ) 3.3 W92A ⁄ L125A ⁄ V127A 19.8 ± 0.3 3.69 ± 0.02 3.59 ± 0.07 ) 4.2 L125F ⁄ V127F 24.9 ± 0.4 3.44 ± 0.08 5.26 ± 0.09 0.9 a DG U H 2 O was determined by extrapolation of the data to a concentration of 0 M urea during denaturation. b m is the slope of the linear dena- turation plot, dDG U ⁄ d[urea]. c [Urea] 50% is the concentration of urea at which 50% of the protein is unfolded. d Values obtained from Eqn (5). Role of small exterior hydrophobic cluster in KSI Y. S. Yun et al. 2002 FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS caused a decrease in the intensity of this peak owing to the quenching of the fluorescence in the cavity of the active site, but no shift of the wavelength at which the spectral intensity is highest (k max ) was observed. Fluorescence intensity at 363 nm was analyzed as a function of the enzyme concentration (Fig. 4). The K D value of d-equilenin for the WT enzyme was found to be 2.00 lm by fitting the data to Eqn (11) (Table 4). Removal of the hydrophobic moieties from the SEHC increased the K D value for d-equilenin by 2.20-, 5.40- and 2.95-fold in W92A, L125A ⁄ V127A and W92A⁄ L125A ⁄ V127A, respectively, suggesting that the hydro- phobic moieties may be important for the enzyme to bind d-equilenin. In L125F ⁄ V127F, the small increase in K D indicates that the addition of hydrophobic moi- eties, such as phenylalanines, does not significantly affect steroid binding. Effect of mutations on 17b-hydroxy-4-estren- 3-one (19-nortestosterone) binding The K D value for 19-nortestosterone was determined by analyzing the changes in UV absorption spectra upon binding 19-nortestosterone to the enzyme. From spectral titration at various steroid concentrations, the K D value of 19-nortestosterone was obtained for each enzyme according to the relationship given in Eqn (12). The spectral titration for the enzymes is shown in Fig. 5 and the calculated K D values are listed in Table 4. The K D value was determined to be 7.28 lm for the WT enzyme. The K D values for W92A, L125A ⁄ V127A and W92A ⁄ L125A⁄ V127A were increased by 2.81-, 5.97- and 2.08-fold, respectively, indicating that the SEHC could affect the affinity towards 19-nortestosterone. The 1.18-fold increased K D of L125F ⁄ V127F suggests that the increased bulkiness of the phenylalanines did not drastically interfere with steroid binding. Structural analysis of W92A To explain the decreased stability and the increased K D values of W92A towards steroids on a structural basis, the crystal structure of W92A was determined at 2.1 A ˚ resolution. It belongs to the space group C222 1 with cell dimensions of a ¼ 35.320 A ˚ , b ¼ 95.871 A ˚ and c ¼ 72.970 A ˚ . Crystallographic data and refine- ment statistics are listed in Table 5. The structure of W92A was almost the same as that of the WT enzyme, with an rmsd of 0.46 A ˚ . Two major structural differ- ences were noticeable (Fig. 6). One is that the b-strand, including Ala92 in W92A, deviated outwards relative Fig. 3. Unfolding rate constants (k U ) at various urea concentrations for the wild-type (WT) enzyme (s), and those of the mutants L125F ⁄ V127F (h), W92A (n), W92A ⁄ L125A ⁄ V127A (e ), and L125A ⁄ V127A (· ). Rate constants were measured in units of s )1 . The unfolding process was monitored by measuring the change in the intrinsic fluorescence intensity of the protein. The excitation wavelength was 285 nm and the emission wavelength 325 nm. Table 3. Changes in free energies of the native state (DDG U ) and the transition state (DDG U à ) for unfolding upon mutation of D 5 -3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI). Measurements were carried out at 25 °C and pH 7.0. Enzyme DG à U H 2 Oa (kcalÆmol )1 ) m à U b (kcalÆmol )1 ÆM) DDG U c (kcalÆmol )1 ) DDG à U d (kcalÆmol )1 ) DDG à U ⁄DDG U (in H 2 O) m à U ⁄ m U WT 27.8 0.504 0.147 W92A 26.4 0.504 ) 3.1 ) 1.4 0.451 0.149 L125A ⁄ V127A 26.5 0.549 ) 3.3 ) 1.3 0.393 0.168 W92A ⁄ L125A ⁄ V127A 25.7 0.621 ) 4.2 ) 2.1 0.500 0.168 L125F ⁄ V127F 28.5 0.595 0.9 0.7 0.777 0.172 a DG à U H 2 O was obtained from extrapolation of DG à U to 0 M urea where DG à U was determined from the fit according to the equation: k U ¼ (k B T ⁄ h)Æexp[–DG à U ⁄ RT]. b m à U is the slope of the linear denaturation plot, dDG à U ⁄ d[urea]. c Values obtained from Eqn (5). d Values obtained from Eqn (9). Y. S. Yun et al. Role of small exterior hydrophobic cluster in KSI FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS 2003 to that of the WT enzyme, and the distance between the a-carbons of Trp92 in the WT enzyme and of Ala92 in W92A, was 1.47 A ˚ , suggesting that the b-sheet structure underneath the hydrophobic layer was largely perturbed by the deletion of the bulky indole ring of Trp92. Furthermore, the side-chain of Leu125 in W92A moved towards the hydrophobic cav- ity as a result of the absence of the bulky indole ring of Trp92, and the distance between the c-carbons of Leu125 in the WT enzyme and W92A was measured to be 2.21 A ˚ . In addition to two structural differences, the accessible surface areas of the side-chains of Leu125 and Val127 were increased by 16.8 and 86.5 A ˚ 2 compared with those of the WT enzyme, respectively, indicating that the removal of the bulky side-chain of Trp92 exposed Leu125 and Val127 to sol- vent to a greater extent. Fig. 4. Changes in the fluorescence emission of d-equilenin at 363 nm, with varying enzyme concentration, for the wild-type enzyme (WT) (s), and for the mutants W92A (n), L125A ⁄ V127A (·), W92A ⁄ L125A ⁄ V127A (e), and L125F ⁄ V127F (h). The excitation wavelength was 335 nm. The curves were obtained by fitting the data to Eqn (11). Table 4. Dissociation constants (K D ) on the binding of d-equilenin and 19-nortestosterone to the the wild-type (WT) enzyme and its mutants. Measurements were carried out at 25 °C and pH 7.0. Enzyme K D (lM) d-Equilenin a 19-Nortestosterone b WT 2.0 ± 0.2 7.2 ± 1.5 W92A 4.4 ± 0.3 20.3 ± 4.1 L125A ⁄ V127A 10.8 ± 1.1 > 43 c W92A ⁄ L125A ⁄ V127A 5.9 ± 0.2 15.0 ± 3.2 L125F ⁄ V127F 2.8 ± 0.4 8.5 ± 2.1 a The K D for d -equilenin was obtained in a buffer containing 10 mM potassium phosphate and 5% (v ⁄ v) methanol. b The K D for 19-nor- testosterone was obtained in a buffer containing 50 m M Tris ⁄ HCl and 100 m M sodium chloride. c The lower limit was indicated owing to the very low value of the difference spectrum and the inaccuracy of the K D value. Fig. 5. UV-spectral titrations to measure the dissociation constant of 19-nortestosterone for the enzymes. For each enzyme, a differ- ence spectrum was obtained by subtracting the spectra originated from the steroid and enzyme from that of their mixture. The absorption maximum (272 nm) of the difference spectrum for the wild-type (WT) enzyme (s), and for the mutants W92A (n), L125A ⁄ V127A (·), W92A ⁄ L125A ⁄ V127A (e), and L125F ⁄ V127F (h) was analyzed at different steroid concentrations. The curves were obtained by fitting the data to Eqn (12). Table 5. Crystallographic data and refinement statistics for the mutant enzyme W92A. Resolution (A ˚ )2.1 R sym (%) 7.2 data completeness, F > 1r (%) 90.0 R standard (%) 22.41 R free (%) 26.88 No. of refined atoms Atom ⁄ water 1031 ⁄ 51 Average B factor 22.363 rmsd bond length (A ˚ ) 0.006549 rmsd bond angles (deg) 1.23348 Ramachandran plot (%) Most favored regions 89.4 Additional allowed regions 10.6 Generously allowed regions 0.0 Role of small exterior hydrophobic cluster in KSI Y. S. Yun et al. 2004 FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS Discussion Our study was intended to identify the role of the SEHC (comprising Trp92, Leu125 and Val127) in KSI-PI for conformational stability and steroid bind- ing. The DG H 2 O U values decreased significantly for all the mutants in which the hydrophobic residue of the SEHC was replaced with alanine (W92A, L125A ⁄ V127A and W92A ⁄ L125A⁄ V127A). However, when the hydrophobicity in the SEHC was increased by sub- stituting leucine and valine with phenylalanines, the DG H 2 O U value increased. These results indicate that the SEHC might improve the overall stability by stabil- izing the solvent-exposed b-sheet constituting the sur- face hydrophobic layer. The mutational study on steroid-binding affinity also revealed that the SEHC plays a role in efficient steroid binding. The crystal structure of W92A showed that the W92A mutation disrupts the solvent-exposed b-sheet. Contribution of the SEHC to conformational stability The hydrophobic interaction in the SEHC of KSI-PI was perturbed by replacing the hydrophobic residues with amino acids having smaller or larger hydrophobic side-chains. The 3.1 kcalÆmol )1 decrease in thermody- namic stability of W92A is noteworthy given that amino acid substitution of a surface residue generally does not affect the stability of a protein [35–38]. The decreased stability of L125A ⁄ V127A and increased sta- bility of L125F ⁄ V127F suggest that the hydrophobic packing of the SEHC is important for the conforma- tional stability of KSI-PI. The stability of L125A ⁄ V127A was decreased by 0.9 kcalÆmol )1 upon the additional mutation of W92A. This additional muta- tion could be expected to stabilize the protein because the hydrophobic Trp92 might not be stable in L125A ⁄ V127A. The marginal decrease of the stability suggested that Trp92 in L125A ⁄ V127A could interact with other nearby hydrophobic moieties as a result of the slight change of local conformation. In previous studies, the b-sheet structure underneath the hydropho- bic layer of the thermolysin-like neutral protease of Bacillus stearothermophilus was found to be stabilized by utilizing a hydrophobic residue at the solvent- exposed site [3], and a hydrophobic pocket on the sur- face of neutral protease of B. subtilis could stabilize the protease [2]. Hence, the stabilizing effects of KSI- PI may originate from hydrophobic interaction medi- ated by the SEHC on the protein surface. Hydrophobic clusters or residues have sometimes been found on the surface of b-sheet structure proteins [5,39]. The SEHC in KSI-PI is located on the center of three b -strands (B4, B5 and B7) that are exposed to solvent. Recent studies on the b-sheet structure sugges- ted that a hydrophobic shield protecting the b-sheet structure against invading water molecules could be required to stabilize solvent-exposed b-sheets [2,4,40]. Invading water is critically related to the kinetic stabil- ity of the protein as protein unfolding can be initiated from the solvent-exposed region by the invasion of water. Consistent with this notion, the unfolding rate constant showed a large increase of 133-fold in W92A ⁄ L125A ⁄ V127A compared with the WT enzyme upon increasing the urea concentration up to 7 m.In the crystal structure of W92A, the deletion of the bulky indole ring of Trp92 significantly perturbed the solvent-exposed b-sheet. Given that backbone chain movement by a single amino acid substitution, especi- ally in the case of a surface residue, has rarely been found, the structural perturbation induced by the W92A mutation is notable. In view of the structural change in W92A at the solvent-exposed b-sheet, we may assume that the decreased stability of W92A ori- ginates from the replacement of the bulky hydrophobic moiety of tryptophan, resulting in increased access of the invading water molecules to the b-sheet, ultimately leading to the acceleration of protein unfolding. The hydrophobic interaction of the SEHC in KSI-PI seems to be partially maintained in the transition state during the unfolding process, as judged by the U U val- ues (Table 3). Solvent-exposed regions, including loops, usually exhibit high U U values, close to 1, because the exposed region is usually exposed to solvent in the transition state for the folding process [33,41]. How- ever, the U U values of W92A, L125A ⁄ V127A and W92A ⁄ L125A ⁄ V127A were found to be below 0.5, indicating that over 50% of the hydrophobic inter- Fig. 6. Stereoview of the small exterior hydrophobic cluster (SEHC) in the wild-type (WT) enzyme and in the mutant W92A after super- imposition of the backbone atoms of all residues. Trp92, Leu125, and Val127 are displayed by a ball-and-stick model, and the back- bone of residues 89–98 and 125–127 are drawn in solid lines. The structure of the WT enzyme is shown in light grey, and that of the mutant W92A is shown in dark grey. The superimposition and drawing were carried out by using the program SWISS-PDB VIEWER, Version 3.7 [49]. Y. S. Yun et al. Role of small exterior hydrophobic cluster in KSI FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS 2005 action was maintained in the transition state during the unfolding process. The high U U value of L125F ⁄ V127F seems to be a result of the increased bulkiness caused by the introduced phenyl rings. In this case, the U U value does not seem to properly represent the status of the transition state in folding, because adding new functional groups can make other extraneous interac- tions and cause steric effects in the protein [33,41]. Our results indicate that the SEHC in KSI-PI contributes to the resistance to unfolding, despite its solvent-exposed site. Analysis of the U U value also supports that the SEHC is required to stabilize the surface conformation in KSI-PI, suggesting that the SEHC can play an important role in the unfolding process in concert with the hydrophobic core. Contribution of the SEHC to recognition of steroids Based on the crystal structure, the SEHC comprising Trp92, Leu125 and Val127 is located on the top of the hydrophobic layer of the steroid-binding pocket in KSI-PI (Fig. 1B). The steroid-binding pocket is lined with hydrophobic residues, which contribute to the tight binding of hydrophobic steroids [19]. The affinity of KSI-PI towards steroids was assessed by utilizing two steroids: d-equilenin and 19-nortestosterone [42]. K D values for both steroids increased by over twofold in all the mutants with decreased hydrophobicity in the SEHC (i.e. W92A, L125A ⁄ V127A and W92A ⁄ L125A ⁄ V127A). Consistent with the increased K D values in those mutants, the K M values of 5-AND increased, indicating that the SEHC contributes to the steroid binding in KSI-PI. The decrease of hydrophobicity in the SEHC, destabilizing the overall hydrophobic layer along the binding site of the steroid, could lead to a decrease in affinity towards the steroids 5-AND, 19-nortestosterone and d-equilenin. In the case of L125A ⁄ V127A, the drastic decrease in the affinity to steroids could be a result of disruption of the SEHC, as Trp92 cannot form a hydrophobic cluster without the aliphatic side-chains of Leu125 and Val127. In L125F ⁄ V127F, the slight increase in K D and K M values suggests that replacing leucine and valine with phenyl- alanines cannot increase the binding affinity of steroids. The solvent-exposed hydrophobic residues may con- tribute to hydrophobic substrate- or ligand-binding to the protein. It was reported that the hydrophobic sur- face made by hydrophobic residues could be important for the binding of phospholipids, vitamin D, lipid and cholesterol to their respective proteins. These observa- tions, as well as ours, suggest that solvent-exposed hydrophobic residues seem to interact with their ligands or substrates on the protein surface in the initial binding step. Even if the hydrophobic residues constituting the SEHC do not directly bind steroids, as judged by the crystal structure of KSI-PI in complex with d-equilenin (Fig. 1B), the SEHC seems to indi- rectly affect the binding process of steroids by stabil- izing the surface hydrophobic layer or perhaps by guiding hydrophobic steroids at the top of the hydro- phobic cleft. The bound mode of the steroid in both KSI-PI and W92A is almost identical based on the X-ray crystal data, supporting the fact that the SEHC might play a role in the initial recognition of hydro- phobic steroids rather than the binding itself. In conclusion, the mutational studies on the role of the SEHC in KSI-PI demonstrate that the SEHC con- tributes not only to conformational stability, but also to the binding affinity of steroids, by stabilizing the hydrophobic surface conformation. Our results suggest that the SEHC stabilizes the hydrophobic layer by connecting the solvent-exposed b-strands and helps to bind hydrophobic steroids. It remains to be investi- gated whether SEHC, as a structural motif, can con- tribute to the conformational stability or the binding of hydrophobic ligands in other proteins. Experimental procedures Materials and reagents 5-AND, d-equilenin and 19-nortestosterone were purchased from Steraloids (Newport, RI, USA). Chemicals for buffer solutions were from Sigma (St Louis, MO, USA). Oligonu- cleotides were obtained from Genotech (Daejon, Korea). A QuickChange Site-Directed Mutagenesis Kit was supplied by Stratagene (La Jolla, CA, USA). pKK 223–3 plasmid was from Pharmacia (New York, NY, USA). A Superose 12 gel filtration column was obtained from Amersham Pharmacia Biotech. Site-directed mutagenesis The QuickChange Site-Directed Mutagenesis Kit (Strata- gene) was used for the mutagenesis. All mutagenesis pro- cedues were carried out according to the instructions provided by the supplier. The pKK 223–3 vector, carrying the KSI-PI gene, was used for the mutagenesis with two primers for each mutant: 5¢-CGCGTCGAGATGGTC GCG AACGGCCAGCCCTGT-3¢ and 5¢-ACAGGGCTGGCCG TTC GCGACCATCTCGACGCG-3¢ (W92A); 5¢-TGGAGC GAGGTCAAC TTCAGCTTCCGCGAGCCGCAGTAG-3¢ and 5¢-CTACTGCGGCTCGCG GAAGCTGAAGTTGAC CTCGCTCCA-3¢ (L125F ⁄ V127F); and 5¢-TGGAGCG AGGTCAAC GCCAGCGCGCGCGAGCCGCAGTAG-3¢ Role of small exterior hydrophobic cluster in KSI Y. S. Yun et al. 2006 FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS and 5¢-CTACTGCGGCTCGCGCGCGCTGGCGTTGAC CTCGCTCCA-3¢ (L125A ⁄ V127A); the constructed pKK 223–3 vector carrying the W92A gene was used for the pre- paration of the triple mutant (W92A ⁄ L125A ⁄ V127A) with two primers; 5¢-TGGAGCGAGGTCAAC GCCAGCGCGC GCGAGCCGCAGTAG-3¢ and 5¢-CTACTGCGGCTCGC GC GCGCTGGCGTTGACCTCGCTCCA-3¢; underlined nucleotides represent those changed by point mutations. Recombinant plasmids were introduced into Escherichia coli XL1-Blue supercompetent cell (Stratagene) and purified by use of a QIAprep Spin Miniprep Kit (Qiagen, ValenciaCA, USA). The entire KSI-PI gene was then sequenced to con- firm the desired mutations. Expression and purification of the KSI-PI proteins WT and mutant KSI-PIs were overproduced in E. coli BL21(DE3) utilizing pKK223-3, an expression vector con- taining the respective KSI-PI gene, and purified by deoxych- olate affinity chromatography and Superose 12 gel filtration chromatography, as described previously [26]. The purity of the protein was confirmed by the presence of a single band on an SDS ⁄ PAGE gel stained with Coomassie blue. The protein concentration was determined by utilizing the differ- ence extinction coefficient between tyrosinate and tyrosine at 295 nm, as described previously [43]. The accuracy of the protein concentration was confirmed by the quantitative analysis of the bands on SDS ⁄ PAGE by use of an imaging densitometer (Bio-Rad, Hercules, CA, USA; GS-700) and a software program (molecular analyst ⁄ PC). Steady-state kinetic analysis Catalytic activities of the purified enzymes were determined spectrophotometrically using 5-AND as a substrate, accord- ing to the procedure previously described [42]. Various amounts of the substrate dissolved in methanol were added to a reaction buffer containing 34 mm potassium phosphate and 2.5 mm EDTA, pH 7.0, at 25 ° C. The concentrations of 5-AND used were 12, 35, 58, 82 and 116 lm. The final concentration of methanol was 3.3% (v ⁄ v). The initial reac- tion rate was obtained within 1 or 2 min after the initiation of the enzymatic reaction. The fraction of the substrate converted to the product was below 10% of the substrate applied to the reaction mixture. The reaction was moni- tored by measuring the absorbance at 248 nm by using a spectrophotometer (Shimadzu, Kyoto, Japan; UV-2501 PC). k cat and K M values were determined by utilizing Line- weaver–Burk reciprocal plots. Calculation of accessible surface area Accessible surface areas were calculated based on the atomic coordinates (PDB code, 4TSU) obtained by X-ray crystallography using the program molmol, 2 k2 [44] according to the method described previously [45]. The probe radius for the calculation was 1.4 A ˚ . Equilibrium unfolding Unfolding of the protein was assessed by measuring the molar ellipticity at different urea concentrations. Protein (15 lm) was incubated for at least 48 h in a buffer containing 20 mm potassium phosphate, pH 7.0, 1 mm EDTA, 1 mm dithiothreitol and different concentrations (0–8 m) of urea. A cuvette with a 0.2 cm path length was used for all CD spectral measurements. The ellipticity at 222 nm was recorded and analyzed. The changes in the optical properties of the protein were compared by normalizing each transition curve with the apparent frac- tion of the unfolded form, F U , which was obtained by Eqn (1): F U ¼ðY N À Y Þ=ðY N À Y U Þ; ð1Þ where Y is the observed molar ellipticity at a given urea concentration, and Y N and Y U are the observed values for the native and unfolded forms, respectively, at the same denaturant concentration. Linear extrapolations from these baselines were made to estimate Y N and Y U in the trans- ition region. The equilibrium constant (K U ) and free- energy change (DG U ) for denaturation were determined, according to a two-state model of denaturation, by Eqns (2) and (3): K U ¼ 2P T Á½F 2 U =ð1 À F U Þ ð2Þ and DG U ¼ÀRT Á ln ðK U Þ¼DG H 2 O U À m Á½urea; ð3Þ where P T is the total protein concentration, DG H 2 O U the free-energy change in the absence of urea, and m a measure of the DG U dependence on urea concentration. DG H 2 O U and m values were obtained by fitting urea denaturation curve data to Eqn (4) [46] using a software program (Abelbeck Softwae, kaleidagraph version 3.06): Y ¼ Y N ÀðY N ÀY U ÞÁexp½ðm Á½ureaÀDG H 2 O U Þ=RT Á½f1 þ8P T =exp½ðm Á½ureaÀDG H 2 O U Þ=RTg 1=2 À1=4P T : ð4Þ The difference in the free-energy change for unfolding, DDG U , between WT and each mutant protein was obtained by Eqn (5): DDG U ¼ DG m U À DG U ; ð5Þ where DG U and DG m U are the free-energy changes for the unfolding of WT and mutant proteins, respectively. Y. S. Yun et al. Role of small exterior hydrophobic cluster in KSI FEBS Journal 272 (2005) 1999–2011 ª 2005 FEBS 2007 Kinetic analysis of unfolding The unfolding kinetic experiments for WT and mutant KSI-PIs were performed by use of a spectrofluorometer (Shimadzu RF5000) equipped with a thermostatically con- trolled cell holder. The protein was incubated in a buffer containing 20 mm potassium phosphate, pH 7.0, 1 mm EDTA and 1 mm dithiothreitol. Unfolding reactions were initiated by diluting the protein sample 20-fold into the same buffer with various concentrations of urea at 25 °C. The dead time of manual mixing was % 10 s. The kinetics for unfolding was monitored by measuring the fluorescence intensity at 325 nm after excitation at 285 nm. The final protein concentration was 15 lm. The rate constants for unfolding at each urea concentration were obtained by fit- ting the data to Eqn (6): F t ¼ F 1 þ R½F i Á expðÀk i Á tÞ; ð6Þ where F t and F 1 are the amplitudes at time t and at the final state, F i is the amplitude of the kinetic phase and k i is the rate constant for unfolding. Data fitting was carried out by using the kaleidagraph program. The unfolding rate constants, k U , obtained at different urea concentra- tions, were then analyzed according to Eqn (7), as des- cribed [41]: ln k U ¼ ln k H 2 O U þ m U z Á½urea; ð7Þ where k H 2 O U is the unfolding rate constant in the absence of urea and m U à the dependence of the unfolding rate con- stant on urea concentration. The free energy of activation for the unfolding of KSI-PI was obtained by Eqn (8): DG z U ¼ DG H 2 Oz U À m U z Á½urea; ð8Þ where DG H 2 O U à is the free-energy change for the unfolding transition state in the absence of urea, and m U à represents a measure of the DG U à dependence on urea concentration. DG U à was obtained from the relationship, DG U à ¼ RTln(k B T ⁄ h)–lnk U , where k B , T and h are the Boltzman constant, the experimental temperature and the Plank con- stant, respectively. Analysis of the F U value The changes in free energy of activation for unfolding, DDG U à , between WT and mutant proteins were obtained by Eqn (9): DDG z U ¼ DG zm U À DG z U ð9Þ where DG U à and DG U àm are the free-energy changes of acti- vation for the unfolding of WT and mutant proteins, respectively. The F value of unfolding, F U , is the ratio of the free-energy change determined from the kinetic data to that determined from the urea equilibrium unfolding experi- ment, as described in Eqn (10): U U ¼ DDG z U =DDG U ¼ðDG F À DG z Þ=ðDG F À DG solv Þ; ð10Þ where DG F is the difference of the noncovalent interaction energy between WT and mutant enzymes in the folded states, DGà the difference in the transition states and DG solv the difference in the unfolded states. Determination of K D for d-equilenin Fluorescence quenching upon the binding of equilenin to the enzyme was used to determine the dissociation constant, K D , as described previously [42]. Fluorescence measure- ments were carried out at 25 °C by using a spectroflurome- ter (Shimadzu, RF5000) in a buffer containing 10 mm potassium phosphate and 5% (v ⁄ v) methanol at pH 7.0. A total of 5 lL of the stock solution of d-equilenin was added to 3.0 mL of the buffer, giving a final concentration of 3 lm. Titrations were carried out by adding 6 lL of the enzyme solution to give a total volume of 72 lL. After add- ing the enzyme, the emission spectrum was scanned from 345 nm to 450 nm with an excitation wavelength at 335 nm. After the spectral change caused by the dilution had been corrected, the fluorescence of d-equilenin at the emission maximum (363 nm) for each enzyme concentra- tion was used to calculate the K D for d-equilenin by nonlin- ear least-squares fitting, according to Eqn (11), by using the kaleidagraph program: E t ¼ðF 0 À FÞfK D =ðF À F 1 Þþ½equilenin=ðF 0 À F 1 Þg; ð11Þ where E t is the concentration of total enzyme in the solu- tion, F is the fluorescence intensity, F 0 is the intensity in the absence of enzyme and F 1 is the intensity extrapolated to infinite enzyme concentration. A binding stoichiometry of 1 per subunit was assumed. Determination of K D for 19-nortestosterone The K D for 19-nortestosterone was determined by UV absorption spectrometry, as described previously [42]. The measurements were carried out at 25 °C, using a spectro- photometer (Shimadzu, UV-2501 PC), in an 1.0 cm quartz cuvette with a total volume of 1 mL. The spectra from 320 to 220 nm were obtained in a buffer containing 50 mm Tris ⁄ HCl and 100 mm sodium chloride at pH 7.0. 19-Nortestosterone was added to the enzyme from a 10 mm stock solution containing 20% (v ⁄ v) methanol. The absorption change caused by the increased volume was corrected. Difference spectra were obtained by subtracting the spectra of total steroid and total enzyme from those of their mixture. The changes in absorption (DA) at the respective absorption maxima in the difference spectra were measured as a function of steroid concentration. K D values were determined by fitting the DA plots, with Role of small exterior hydrophobic cluster in KSI Y. S. 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Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype. KSI-PI to stabilize the hydro- phobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic