A structural motif known as the small exterior hydrophobic cluster Keywords conformational stability; ketosteroid isomerase; small exterior hydrophobic cluster; steroid binding; surface
Trang 1conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B
Young S Yun1,2, Gyu H Nam1,2, Yeon-Gil Kim1,3, Byung-Ha Oh1,3and Kwan Y Choi1,2
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 D5-3-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 UU 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.
Trang 2(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]
D5-3-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
D5-3-ketosteroids to D4-3-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 cenclus-ter 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 PI Even though
KSI-PI exposes the hydrophobic residues to solvent, it exhibits high thermodynamic stability (a DG H2 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
H
Tyr14 OH
Asp99
Asp38
Asp99
Asp38
Asp99
Asp38
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.
Trang 3Structure 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 kcat and KM 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 KM more than the kcat The kcat 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 KMvalues 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, DGU, was deter-mined by monitoring the molar ellipticity of the pro-teins at 222 nm upon changing the urea concentration
at 25C 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 H2 O
U , m, and DDGU for wild-type (WT) and mutant enzymes were obtained (Table 2) Removal of hydrophobic moieties from the SEHC decreased the DG H2 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 m M EDTA, pH 7.0.
cata Relative kMb
99.5 ± 23.7 1.3 · 10 8
L125F ⁄ V127F (17.6 ± 0.8) · 10 3
79.5 ± 3.8 2.3 · 10 8
a,b
Values relative to those of the WT enzyme.cData from [42].
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].
Trang 4in W92A and W92A⁄ L125A ⁄ V127A, respectively.
However, the L125F⁄ V127F mutation of the SEHC
increased the DG H2 O
U value by 0.9 kcalÆmol)1, while the L125A⁄ V127A mutation of the SEHC decreased
the DG H2 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 kU 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 UU value analy-sis, according to the method described previously [33] The UUvalue can range from 0 to 1 A high UUvalue 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 UUvalue 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 UU 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 UU value of 0.777 The ratio of
mU⁄ mU has been reported to indicate the increase in solvent exposure of the transition state relative to the native state [34] The mU⁄ mU 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 post-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 2Oa (kcalÆmol)1)
m b (kcalÆmol)1Æ M )
[Urea] 50%c ( M )
DG Ud (kcalÆmol)1)
a DG U 2Owas determined by extrapolation of the data to a concentration of 0 M urea during denaturation.bm 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).
Trang 5caused 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 (kmax) was observed
Fluorescence intensity at 363 nm was analyzed as a
function of the enzyme concentration (Fig 4) The KD
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 KD 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 KDindicates 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 KD 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
KDvalue 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 KD values are listed in Table 4 The KD value was determined to be 7.28 lm for the WT enzyme The KDvalues 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 KD 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
KD 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 C2221 with cell dimensions of a¼ 35.320 A˚, b ¼ 95.871 A˚ and c¼ 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) 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
U 2Oa (kcalÆmol)1)
mUb (kcalÆmol)1Æ M )
DDG Uc (kcalÆmol)1)
DDG
Ud (kcalÆmol)1)
DDG
U ⁄ DDG U
U ⁄ m U
a DG
U 2Owas obtained from extrapolation of DG
U to 0 M urea where DG
U was determined from the fit according to the equation: kU¼ (kBT ⁄ h)Æexp[–DG
U ⁄ RT] b m
U is the slope of the linear denaturation plot, dDG
U ⁄ d[urea] c Values obtained from Eqn (5) d Values obtained from Eqn (9).
Trang 6to 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 (KD) 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
KD(l M ) d-Equilenina 19-Nortestosteroneb
L125A ⁄ V127A 10.8 ± 1.1 > 43c
W92A ⁄ L125A ⁄ V127A 5.9 ± 0.2 15.0 ± 3.2
a The KDfor d-equilenin was obtained in a buffer containing 10 m M
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 KDvalue.
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.
No of refined atoms
Ramachandran plot (%)
Trang 7Our 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 H2 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 H2 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 UU val-ues (Table 3) Solvent-exposed regions, including loops, usually exhibit high UU 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 UU 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].
Trang 8action was maintained in the transition state during the
unfolding process The high UUvalue of L125F⁄ V127F
seems to be a result of the increased bulkiness caused
by the introduced phenyl rings In this case, the UU
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 UU 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]
KD 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 KD values in
those mutants, the KM 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 KDand KMvalues
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¢-CGCGTCGAGATGGTCGCG AACGGCCAGCCCTGT-3¢ and 5¢-ACAGGGCTGGCCG TTCGCGACCATCTCGACGCG-3¢ (W92A); 5¢-TGGAGC GAGGTCAACTTCAGCTTCCGCGAGCCGCAGTAG-3¢ and 5¢-CTACTGCGGCTCGCGGAAGCTGAAGTTGAC CTCGCTCCA-3¢ (L125F⁄ V127F); and 5¢-TGGAGCG AGGTCAACGCCAGCGCGCGCGAGCCGCAGTAG-3¢
Trang 9and 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¢-TGGAGCGAGGTCAACGCCAGCGCGC
GCGAGCCGCAGTAG-3¢ and 5¢-CTACTGCGGCTCGC
GCGCGCTGGCGTTGACCTCGCTCCA-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 25C 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 initiareac-tion
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) kcatand KMvalues 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, FU, which was obtained by Eqn (1):
FU¼ ðYN YÞ=ðYN YUÞ; ð1Þ
where Y is the observed molar ellipticity at a given urea concentration, and YNand YUare the observed values for the native and unfolded forms, respectively, at the same denaturant concentration Linear extrapolations from these baselines were made to estimate YN and YU in the trans-ition region The equilibrium constant (KU) and free-energy change (DGU) for denaturation were determined, according to a two-state model of denaturation, by Eqns (2) and (3):
KU¼ 2PT ½F 2
U=ð1 FUÞ ð2Þ and
DGU¼ RT ln ðKUÞ ¼ DG H 2 O
U m ½urea; ð3Þ where PT is the total protein concentration, DG H2 O
free-energy change in the absence of urea, and m a measure
of theDGUdependence on urea concentration DG H2 O
m values were obtained by fitting urea denaturation curve data to Eqn (4) [46] using a software program (Abelbeck Softwae, kaleidagraph version 3.06):
Y¼ YN ðYN YUÞ exp½ðm ½urea DGH 2 O
U Þ=RT
½f1 þ 8PT=exp½ðm ½urea DGH2 O
U Þ=RTg1=2 1=4PT:
ð4Þ The difference in the free-energy change for unfolding, DDGU, between WT and each mutant protein was obtained
by Eqn (5):
DDGU¼ DG m DGU; ð5Þ where DGU and DG mare the free-energy changes for the unfolding of WT and mutant proteins, respectively
Trang 10Kinetic 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 25C
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):
Ft¼ F1þ R½Fi expðki tÞ; ð6Þ
where Ft and F1 are the amplitudes at time t and at the
final state, Fiis the amplitude of the kinetic phase and ki
is the rate constant for unfolding Data fitting was carried
out by using the kaleidagraph program The unfolding
rate constants, kU, obtained at different urea
concentra-tions, were then analyzed according to Eqn (7), as
des-cribed [41]:
ln kU¼ ln kH 2 O
U þ mU z ½urea; ð7Þ where k H2 O
U is the unfolding rate constant in the absence of
urea and mU 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):
DGzU¼ DG H2 Oz
U mU z ½urea; ð8Þ where DG H2 O
U
is the free-energy change for the unfolding
transition state in the absence of urea, and mUrepresents
a measure of the DGU dependence on urea concentration
DGU was obtained from the relationship, DGU¼
RTln(kBT⁄ h) – lnkU, where kB, T and h are the Boltzman
constant, the experimental temperature and the Plank
con-stant, respectively
Analysis of theFUvalue
The changes in free energy of activation for unfolding,
DDGU, between WT and mutant proteins were obtained by
Eqn (9):
DDGUz¼ DGUzm DGUz ð9Þ
whereDGUandDGUmare the free-energy changes of
acti-vation for the unfolding of WT and mutant proteins,
respectively The F value of unfolding, FU, 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):
UU¼ DDGzU=DDGU¼ ðDGF DGzÞ=ðDGF DGsolvÞ;
ð10Þ where DGFis the difference of the noncovalent interaction energy between WT and mutant enzymes in the folded states,DG the difference in the transition states and DGsolv
the difference in the unfolded states
Determination of KDfor d-equilenin Fluorescence quenching upon the binding of equilenin to the enzyme was used to determine the dissociation constant,
KD, as described previously [42] Fluorescence measure-ments were carried out at 25C 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 KDfor d-equilenin by nonlin-ear least-squares fitting, according to Eqn (11), by using the kaleidagraphprogram:
Et¼ ðF0 FÞfKD=ðF F1Þ þ ½equilenin=ðF0 F1Þg; ð11Þ where Et is the concentration of total enzyme in the solu-tion, F is the fluorescence intensity, F0is the intensity in the absence of enzyme and F1 is the intensity extrapolated to infinite enzyme concentration A binding stoichiometry of
1 per subunit was assumed
Determination of KDfor 19-nortestosterone The KD for 19-nortestosterone was determined by UV absorption spectrometry, as described previously [42] The measurements were carried out at 25C, 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 KD
values were determined by fitting the DA plots, with