peptidyl prolyl cis-trans isomeraseImportance of a V-shaped dimeric structure for binding to protein substrate Cahyo Budiman1, Keisuke Bando1, Clement Angkawidjaja1, Yuichi Koga1, Kazufu
Trang 1peptidyl prolyl cis-trans isomerase
Importance of a V-shaped dimeric structure for binding to protein substrate
Cahyo Budiman1, Keisuke Bando1, Clement Angkawidjaja1, Yuichi Koga1, Kazufumi Takano1,2and Shigenori Kanaya1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka, Japan
2 CRESTO, JST, Yamadaoka, Suita, Osaka, Japan
Keywords
FKBP22; homodimer; peptidyl-prolyl
cis-trans isomerase (PPIase); protein
engineering; substrate binding
Correspondence
S Kanaya, Department of Material and Life
Science, Graduate School of Engineering,
Osaka University, 2-1, Yamadaoka, Suita,
Osaka 565-0871, Japan
Tel ⁄ Fax: +81 6 6879 7938
E-mail: kanaya@mls.eng.osaka-u.ac.jp
(Received 1 May 2009, revised 24 May
2009, accepted 28 May 2009)
doi:10.1111/j.1742-4658.2009.07116.x
FK506-binding protein 22 (FKBP22) from the psychrotrophic bacterium Shewanellasp SIB1 is a homodimeric protein with peptidyl prolyl cis–trans isomerase (PPIase) (EC 5.2.1.8) activity Each monomer consists of 205 amino acid residues According to a tertiary model, SIB1 FKBP22 assumes
a V-shaped structure, in which two monomers interact with each other at their N-termini Each monomer consists of an N-terminal domain with a dimerization core and a C-terminal catalytic domain, which are separated
by a 40-residue-long a-helix To clarify the role of this V-shaped structure,
we constructed a mutant protein, in which the N-domain is tandemly repeated through a flexible linker This protein, termed NNC-FKBP22, is designed such that two repetitive N-domains are folded into a structure similar to that of the Shewanella sp SIB1 FKBP22 wild-type protein (WT) NNC-FKBP22 was overproduced in Escherichia coli in a His-tagged form, purified and biochemically characterized Gel-filtration chromatography and ultracentrifugation analyses indicate that NNC-FKBP22 exists as a monomer Analysis of thermal denaturation using differential scanning cal-orimetry indicates that NNC-FKBP22 unfolds with two transitions, as does the WT protein NNC-FKBP22 exhibited PPIase activity for both peptide and protein substrates However, in contrast to its activity for peptide sub-strate, which was comparable to that of the WT protein, its activity for protein substrate was reduced by five- to six-fold, compared to that of the
WT Surface plasmon resonance analyses indicate that NNC-FKBP22 binds to a reduced form of a-lactalbumin with a six-fold weaker affinity than that of WT These results suggest that a V-shaped structure of SIB1 FKBP22 is important for efficient binding to a protein substrate
Structured digital abstract
l MINT-7136140 : FKBP22 (uniprotkb: Q765B0 ) binds ( MI:0407 ) to Alpha-lactalbumin (uni-protkb: P00711 ) by surface plasmon resonance ( MI:0107 )
Abbreviations
DSC, differential scanning calorimetry; FKBP, FK506-binding protein; MIP, macrophage-infectivity potentiator; pNA, p-nitroanilide; PPIase, peptidyl prolyl cis–trans isomerase; RCM, reduced and carboxymethylated; suc-ALPF-pNA, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; WT, Shewanella sp SIB1 FKBP22 wild-type protein.
Trang 2Peptidyl prolyl cis-trans isomerase (PPIase) (EC
5.2.1.8) catalyzes cis–trans isomerization of the Xaa–
Pro peptide bonds of proteins [1] Because the peptide
bond in the cis conformation is energetically
unfavor-able compared with that in the trans conformation
[2,3], and cis–trans isomerization of this peptide bond
is intrinsically very slow [4], prolyl isomerization is
regarded as a rate-limiting step of the folding reaction
of proteins that contain cis prolines in a folded state
[5] PPIases are divided into four structurally unrelated
families: FK506-binding proteins (FKBPs),
cyclophi-lines, parvulins and the Ser⁄ Thr phosphatase 2A
activator, PTPA [6]
FKBP22 is a member of the FKBP family and
pres-ent in various Gram-negative bacteria [7–9] It is a
homodimer, in which each subunit consists of an
N-terminal domain (N-domain) and a C-terminal
PPIase domain (C-domain) Based on its similarities to
the macrophage-infectivity potentiator (MIP) protein
from Legionella pneumophila in amino acid sequence,
FKBP22 has been classified as a member of the
MIP-like FKBP subfamily [7] Escherichia coli FkpA is also
a member of this subfamily [10] Of the members of
this subfamily, L pneumophila MIP [11] and E coli
FkpA [12] are the only ones for which the crystal
structures have been determined These structures
strongly resemble each other, having an rmsd of 0.8 A˚
for all Ca atoms According to these structures, these
proteins assume a nonglobular V-shaped homodimeric
structure, in which two monomers interact with each
other at their N-domains Each monomer assumes a
dumbbell-like structure, in which the N-domain
(con-sisting of a1 and a2 helices) and the C-domain
[con-sisting of six b strands (b1–b6) and an a4 helix] are
linked by a 40-residue-long a3 helix As a result, the
C-domains, which are located at both ends of a
V-shaped structure, face each other across the cleft of
this structure The interface of two monomers, which
is located at the bottom of the V-shaped structure, is
stabilized by the hydrophobic interactions between the
a1 helix of one monomer and the a2 helix of the other
However, the role of a V-shaped structure of MIP-like
FKBP subfamily proteins remains to be understood
We have previously shown that FKBP22 from the
psychrotrophic bacterium Shewanella sp SIB1 exists
as a homodimer and exhibits PPIase activity for both
peptide and protein substrates [8] SIB1 FKBP22
shows amino acid sequence identities of 56% to
E coli FKBP22 [7], of 43% to E coli FkpA [10] and
of 41% to L pneumophila MIP [13] Construction of
the mutant proteins N-domain+ and C-domain+,
which lack the C- and N-domains of SIB1 FKBP22, respectively, followed by biochemical characterization
of these proteins, suggest that the C-domain is required for PPIase activity, and the N-domain is required for dimerization and efficient binding to a protein substrate of PPIase [14,15] However, it remains to be determined whether a V-shaped struc-ture is required for efficient binding of SIB1 FKBP22
to a protein substrate, because the N-domain+, which retains the ability to bind to a protein substrate, con-tains the entire a3 helix and therefore may be able to assume a V-shaped homodimeric structure Attempts
to construct the N-domain without the a3 helix have
so far been unsuccessful because of the instability of the protein
In this report, we constructed the mutant protein, NNC-FKBP22, in which the N-terminal region (Met8– Ala60) is repeated twice within a single polypeptide chain, and characterized it biochemically This mutant protein was designed such that the repetitive N-termi-nal region is folded into a structure similar to that of the Shewanella sp SIB1 FKBP22 wild-type protein (WT), which has a homodimeric structure Based on these results, we discuss the role of a V-shaped struc-ture of FKBP22
Results Design of monomeric mutant protein The monomeric mutant protein (NNC-FKBP22) was designed based on a model of the 3D structure of WT (SIB1 FKBP22) (Fig 1A), which has previously been reported [14] According to this model, WT assumes a V-shaped homodimeric structure, like those of L pneu-mophila MIP [11] and E coli FkpA [12] In this struc-ture, the Ala60 of one monomer is located in close proximity to the Met8 of the other monomer Both residues are located close to the bottom of the cleft of the V-shaped structure Therefore, it is strongly expected that the mutant protein, termed NNC-FKBP22, in which Met1–Ala60 of SIB1 FKBP22 is attached to Met8–Ile205 of SIB1 FKBP22 through three glycine residues, is monomeric and folded into a structure similar to that of WT without one arm of the
‘V’ A model of the 3D structure of NNC-FKBP22 is shown in Fig 1A Its primary structure is also sche-matically shown in Fig 1B in comparison with that of
WT NNC-FKBP22 and SIB1 FKBP22 (WT) in a His-tagged form will be designated as NNC-FKBP22 and SIB1 FKBP22 (WT), respectively, hereafter
Trang 3NNC-FKBP22 was overproduced in E coli at
10C, as previously reported for WT [8] The protein
accumulated in the E coli cells in a soluble form and
was purified to give a single band on SDS–PAGE (see,
Fig S1) WT was also overproduced and purified as
reported previously [8]
Determination of oligomeric state
The molecular mass values of NNC-FKBP22 and WT
were estimated to be 34 and 28 kDa, respectively, from
SDS–PAGE These values are considerably higher than
those calculated from their amino acid sequences
(27 kDa for NNC-FKBP22 and 21 kDa for WT) It
has been reported for WT that the molecular mass of
the protein determined by ESI-MS (23 kDa) is
compa-rable to the calculated molecular mass and therefore
the molecular mass of the protein estimated from
SDS–PAGE is considerably higher than the calculated
molecular mass as a result of its unusual behavior on
SDS–PAGE [8] Because the difference in molecular
mass values of NNC-FKBP22 and WT, estimated
from SDS–PAGE, is comparable to the difference in
their molecular mass values calculated from amino acid sequences, the molecular mass of NNC-FKBP22
is considerably higher than the calculated molecular mass, probably as a result of its unusual behavior on SDS–PAGE, like WT The molecular mass values of native forms of NNC-FKBP22 and WT were estimated
to be 58 and 99 kDa, respectively, from gel-filtration column chromatography These values are 4.5- and 2.1-fold higher than those calculated from their amino acid sequences However, it has been reported for WT that the molecular mass of the protein, as determined
by sedimentation equilibrium analytical ultracentrifu-gation (44 kDa), is two-fold higher than the calculated molecular mass, and the discrepancy between the molecular mass values estimated from gel filtration and analytical ultracentrifugation is a result of the unusual behavior of WT on gel filtration The unusual behavior of the protein on gel filtration has also been reported for L pneumophila MIP The molecular mass
of this protein, as estimated from gel filtration, is higher than the calculated molecular mass by 2.7-fold, instead of by two-folds, because it is cylindrical rather than globular [16] Therefore, the molecular mass
9 23 34 45 52 93
95 99
102 111
121 130
134 139
146 150
155 163
167 174
195 205
α1 α2 α3 β1 β2 β3 β4a β4b α4 β5 β6
1
N-domain
B
C-domain
65 79 90 101 108 149
151 155
158 167
177 186
190 195
202 206
211 219
223 230
251 261
α4 α5 α6 β1 β2 β3 β4a β4b α7 β5 β6
9 23 34 45 52
α1 α2 α3
1
60
GGG
C-domain N-domain
SIB1 FKBP22
NNC-FKBP22
C-domain
Ala60
(Gly) 3 Met64
N-domain
N-domain
C-domain C-domain
Ala60 Met8
α1
α5 α4
α2 α2
α1
α2
α1
α3
A
Fig 1 (A) 3D structure models of SIB1
FKBP22 and NNC-FKBP22 For the SIB1
FKBP22 structure, one monomer is
deeply-colored, while the other is lightly-colored.
The N- and C-domains and the a1-3 helices
are indicated The side chain of Met8 of one
monomer and that of Ala60 of the other
monomer are indicated by stick models For
the NNC-FKBP22 structure, the
correspond-ing domains, helices and side chains of the
amino acid residues are indicated A loop
consisting of three glycine residues, which
connects Ala60 and Met64 (corresponding
to Met8 of SIB1 FKBP22), is schematically
shown in cyan (B) Schematic
representa-tions of the primary structures of SIB1
FKBP22 and NNC-FKBP22 A His-tag
attached to the N-termini of the proteins is
represented by a shaded box The a-helices
and b-strands are represented by cylinders
and arrows, respectively These secondary
structures are arranged based on tertiary
models of SIB1 FKBP22 and NNC-FKBP22.
Numbers indicate the positions of the
dues relative to the initiator methionine
resi-due of the proteins without a His-tag The
ranges of the N- and C-domains are also
shown.
Trang 4values of native forms of NNC-FKBP22 and WT were
also determined using sedimentation equilibrium
analytical ultracentrifugation The data fitted well to
a single-species model, and showed no evidence of
aggregation, and the molecular mass values of
NNC-FKBP22 and WT were determined as 30.4 and
43.9 kDa, respectively These values are 1.1- and
1.9-fold larger than the calculated molecular mass
values, indicating that NNC-FKBP22 and WT exist as
a monomer and a dimer in solution, respectively
CD spectra
The far- and near-UV CD spectra of NNC-FKBP22
and WT were measured at 10C The far-UV CD
spectrum of NNC-FKBP22 is similar to that of WT
(Fig 2A) However, the depth of its trough is slightly
larger than that of WT From these spectra, the helical
contents of NNC-FKBP22 and WT are estimated to
be 44 and 38%, respectively, using the method of Wu
et al [17], which are comparable to those calculated
from their tertiary models (54% for NNC-FKBP22
and 49% for WT) The near-UV CD spectrum of
NNC-FKBP22 is also similar to that of WT, although the height of its peak is slightly larger than that of WT (Fig 2B) The near-UV CD spectra reflect the 3D environments of aromatic residues, such as Tyr and Trp WT contains one Trp residue and seven Tyr resi-dues in each monomer This tryptophan residue (Trp157) is conserved in the C-domain of MIP-like FKBP subfamily proteins and is required for PPIase activity In addition, six of the seven Tyr residues are located in the C-domain Therefore, the near-UV CD spectra of these proteins mainly reflect the conforma-tion of the C-domain These results suggest that the 3D structure of NNC-FKBP22 is similar to that of
WT, except that the a3 helix and C-domain of one monomer are removed
Thermodynamics of unfolding SIB1 FKBP22 is thermally denatured with two transi-tions, the first and the second ones for denaturation of the C- and N-domains, respectively [14] To examine whether NNC-FKBP22 gives a similar thermal-dena-turation curve, heat-induced unfolding of NNC-FKBP and WT was analyzed by differential scanning calorim-etry (DSC) Thermal unfolding of these proteins was highly reversible, as indicated by repeating thermal scans to reproduce DSC curves Both proteins gave denaturation curves with two well-separated transitions (Fig 3), suggesting that both domains of NNC-FKBP22 are folded into structures similar to those of
WT However, both domains of NNC-FKBP22 are apparently more stable than those of WT Deconvolu-tion of the thermogram of NNC-FKBP22, according
to a non two-state denaturation model, gives melting temperature (Tm) values of 36.9 and 50.3C for the
2 ·dmol
Wavelength (nm) –20 000
–15 000
–10 000
–5000
0
5000
A
B
Wavelength (nm) –400
–200
0
200
400
600
Fig 2 CD spectra of NNC-FKBP22 The far-UV (A) and near-UV (B)
CD spectra of NNC-FKBP22 (thick line) are shown in comparison
with those of SIB1 FKBP22 (thin line) Both spectra were measured
at 10 C as described in the Experimental procedures.
Temperature (°C) 0
5 10
15 20 25
Fig 3 DSC curve of FKBP22 The DSC curve of NNC-FKBP22 (thick line) is shown in comparison with that of SIB1 FKBP22 (thin line) These curves were measured at a scan rate of 1CÆmin)1 Both proteins were dissolved in 20 m M sodium phos-phate (pH 8.0) at approximately 1 mgÆmL)1.
Trang 5first and second transitions, which probably reflect
denaturation of the C- and N-domains, respectively
These values are higher than the corresponding values
of WT by approximately 4C (Table 1)
PPIase activity
The PPIase activitiy for peptide substrate was
deter-mined using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide
(Suc-ALPF-pNA) as a substrate The catalytic
efficien-cies (kcat⁄ Km) of NNC-FKBP22 and for each
0.04 lm)1Æs)1, respectively, indicating that the catalytic
efficiency of NNC-FKBP22 is slightly higher than, but
comparable to, that of WT The temperature
depen-dence of the PPIase activity of NNC-FKBP22 was
almost identical to that of WT (Fig 4A) By contrast,
when the PPIase activity was determined by a
refold-ing assay usrefold-ing RNase T1 as a protein substrate,
NNC-FKBP22 exhibited much lower activity than that
of WT RNase T1 has been widely used as a protein
substrate for PPIase activity, because cis–trans
isomeri-zation of two peptidyl prolyl bonds (Tyr38–Pro39 and
Ser54–Pro55) of RNase T1is a rate-limiting step of its
folding [18–20] The refolding of RNase T1 was not
seriously accelerated in the presence of 10 nm
NNC-FKBP22 (Fig 4B), while it was significantly
acceler-ated in the presence of 75 nm NNC-FKBP22 to a level
similar to that observed in the presence of 10 nm WT
(data not shown) The kcat⁄ Km values of
NNC-FKBP22 and WT were estimated to be 0.08 ± 0.005
and 0.53 ± 0.03 lm)1Æs)1, respectively
Binding to reduced a-lactalbumin
a-Lactalbumin is stabilized by four disulfide bonds and
a single Ca2 +ion [21,22], and therefore reduction of
these disulfide bonds produces a protein with a
par-tially folded molten globule-like structure [23]
Reduced and carboxymethylated (RCM) a-lactalbumin
has been used as a folding intermediate of proteins to
analyze the chaperone functions of GroEL [24,25] and FKBP family proteins [15,26,27] RCM a-lactalbumin has been shown to compete with the protein substrate
of PPIase for binding to the trigger factor [26] and
E coli FkpA [27], suggesting that RCM a-lactalbumin and a protein substrate of PPIase share a common binding site of FKBP family proteins In order to examine whether NNC-FKBP22 binds to a protein substrate with similar affinity as that of WT, the binding affinities of NNC-FKBP22 and WT to reduced a-lactalbumin were analyzed using surface plasmon resonance (Biacore) Reduced a-lactalbumin was injected onto the sensor chip, on which NNC-FKBP22
or WT was immobilized The amount of protein
Table 1 Thermodynamic parameters for heat-induced unfolding of
the protein The melting temperature (T m ), calorimetric enthalpy
(DHcal) and van’t Hoff enthalpy (DHvH) of SIB1 FKBK22 and
NNC-FKBP22 were obtained from the DSC curves shown in Fig 3 using
ORIGIN software (Microcal Inc.)
RNase H Transition Tm(C)
DH cal
(kJÆmol)1)
DH vH
(kJÆmol)1) SIB1 FKBP22 First 32.5 ± 0.13 83 ± 2.2 404 ± 4.1
Second 46.4 ± 0.07 195 ± 2.1 304 ± 2.0
NNC-FKBP22 First 36.9 ± 0.04 143 ± 1.4 305 ± 3.5
Second 50.3 ± 0.02 250 ± 1.5 327 ± 2.1
Temperature (°C)
0
0.5
Kcat
1
1.5
A
B
Time (s)
40
60
80
100
Fig 4 PPIase activities of NNC-FKBP22 (A) The temperature dependence of the PPIase activity of NNC-FKBP22 (closed circle), which was determined by a protease-coupling assay using Suc-ALPF-pNA as a substrate, is shown in comparison with that of SIB1 FKBP22 (open circle) The catalytic efficiency was calculated according to Harrison & Stein [46] The experiment was carried out
in duplicate Each plot represents the average value, and errors from the average values are shown (B) The increase of tryptophan fluorescence at 323 nm during the refolding of RNase T 1 (0.2 l M )
is shown as a function of the refolding time Refolding was carried cout at 10 C in the absence (broken line), or presence of 10 n M of NNC-FKBP22 (thick solid line) or SIB1 FKBP22 (thin solid line).
Trang 6immobilized on the sensor chip was equivalent to 1200
resonance units for NNC-FKBP22 and 4000 resonance
units for WT The sensorgrams obtained by injecting
100 lm of reduced a-lactalbumin onto these sensor
chips are shown in Fig 5A as a representative
Because the association and dissociation of reduced
a-lactalbumin were too fast to determine the kinetic
constants, such as konand koff, accurately, the
dissoci-ation constant, KD, was determined by measuring
equi-librium-binding responses at various concentrations of
reduced a-lactalbumin The plots of the
equilibrium-binding responses as a function of the concentration of
a-lactalbumin gave a saturation curve, as shown in Fig 5B These plots showed a good fit to a single binding-affinity model and the KDvalue for binding of reduced a-lactalbumin to NNC-FKBP22 was deter-mined to be 42.5 ± 2.1 lm This value is higher than that of WT (6.5 ± 0.38 lm) by 6.5-fold, indicating that the binding affinity of NNC-FKBP22 to a folding inter-mediate of proteins, and probably to a protein substrate,
is greatly reduced compared with that of WT
Discussion Role of a V-shaped structure
In this report, we showed that NNC-FKBP22 is mono-meric and that its binding affinity to reduced a-lactal-bumin and its PPIase activity for protein substrate are reduced by five- to six-fold compared with those of
WT These results strongly suggest that a V-shaped structure of the SIB1 FKBP22 homodimer is impor-tant for binding to a folding intermediate of proteins and therefore for PPIase activity for a protein sub-strate Neither the NNC-FKBP22 nor the WT struc-ture has been determined However, because of the high amino acid sequence similarity between SIB1 FKBP22 and E coli FkpA [10] or L pneumophila MIP [13], SIB1 FKBP22 might assume a V-shaped homodi-meric structure such as E coli FkpA [12] and L pneu-mophila MIP [11] Hu et al [28] have proposed a
‘Mother’s arm’ model for the substrate-binding mecha-nism of E coli FkpA, which exhibits both PPIase and chaperone activities, based on the observation that the a3 helix is rather flexible and controls plasticity of a V-shaped structure According to this model, two long a3 helices act as flexible ‘arms’, which can bend at the
‘elbows’ (presumably located at the middle of the a3 helix) Two catalytic domains act as ‘hands’ and the active-site residues act as ‘fingers’ for protein sub-strates As ‘mother’ holds her ‘baby’ by bending both
of her arms, a dimer form of E coli FkpA holds a protein substrate by bending its two long a3 helices A V-shaped structure of SIB1 FKBP22 may also be required to hold a protein substrate with a similar mechanism The plasticity of a V-shaped structure may lead to a conformational flexibility to adopt various types of protein substrates
The importance of a V-shaped dimeric structure for binding various types of protein substrates has also been reported for a protein disulfide isomerase, DsbC, from E coli [29] E coli DsbC is a homodimer of the 23-kDa protein and assumes a V-shaped structure The structural arrangement of E coli DsbC is similar to those of E coli FkpA and L pneumophila MIP, and
Time (s)
0
200
400
600
A
B
0 20 40 60
0
100
200
300
400
500
600
(Reduced α-lactalbumin) (μ M )
Fig 5 Binding of reduced a-lactalbumin to NNC-FKBP22 and to
SIB1 FKBP22 (A) Sensorgrams from Biacore X showing the binding
of reduced a-lactalbumin (100 l M ) to immobilized NNC-FKBP22
(thick line) and SIB1 FKBP22 (thin line) The sensorgram showing
the binding of nonreduced a-lactalbumin (100 l M ) to NNC-FKBP22,
which is similar to that to SIB1 FKBP22, is also shown (broken
line) Injections were performed at time zero for 60 s (B)
Relation-ships between the equilibrium-binding response and the
concentra-tion of reduced a-lactalbumin The equilibrium-binding responses of
NNC-FKBP22 (closed circle) and of SIB1 FKBP22 (open circle) are
shown as a function of the concentration of reduced a-lactalbumin.
The solid line represents the fitting curve of a single binding-site
affinity model using the BIAevaluation program.
Trang 7its N- and C-domains are connected by a hinged
three-turn linker helix A characteristic common to these
proteins is that the cleft of a V-shaped structure is
more hydrophobic than it is externally The cleft of a
V-shaped structure of SIB1 FKBP22 is also more
hydrophobic than it is externally, suggesting that SIB1
FKBP22 binds to a protein substrate mainly through
hydrophobic interactions
It has been reported that slyD proteins [30–32], an
archaeal FKBP17 [33,34] and FKBP12 with a
heterol-ogous chaperone domain [35], all of which are FKBP
family proteins, exhibit PPIase activities for both
pep-tide and protein substrates when in a monomeric form
The findings from the present study, that
NNC-FKBP22 with a monomeric structure exhibits PPIase
activities for these substrates, is consistent with these
results However, the role of a dimeric structure of
MIP-like FKBP subfamily proteins has not so far been
analyzed It has been reported that the PPIase activity
of the E coli FkpA mutant, which lacks the N-domain
and therefore loses the ability to form a dimeric
struc-ture, is indistinguishable from that of the wild-type
protein [12,36] However, the C-domain of E coli
FkpA tends to oligomerize [36] and therefore the role
of a V-shaped dimeric structure of E coli FkpA
can-not be clearly understood by analyzing this mutant
protein Thus, this is the first report which shows the
importance of a V-shaped dimeric structure of an
MIP-like FKBP subfamily protein for binding to a
protein substrate However, it remains to be
deter-mined whether SIB1 FKBP22 exhibits a chaperone
function and, if so, whether its dimeric structure is
responsible for this function
PPIase activities of NNC-FKBP22
NNC-FKBP22 exhibits 1.4-fold higher activity than
WT (per monomer) for a peptide substrate It has
pre-viously been shown that the mutant protein of SIB1
FKBP22 (C-domain+), which lacks the N-domain and
exists as a monomer, exhibits 1.6-fold higher activity
than WT (per monomer) [14] These results suggest
that the C-domain is sufficient for the binding and
catalysis of a peptide substrate These results also
sug-gest that a V-shaped structure is not favorable for
binding a peptide substrate In this structure, freedom
of each catalytic domain is probably restricted and
therefore the opportunity of this domain to contact
with the substrate decreases By contrast, in a
mono-meric structure, the freedom of the catalytic domain
increases and therefore the opportunity of this domain
to contact with the substrate increases By contrast,
for a protein substrate, the activity of NNC-FKBP22
is six-folds lower than that of WT (per monomer) In this case, only one of the two catalytic domains of WT may serve as a catalytic site because the space between them seems to be too small to accommodate two pro-tein substrates simultaneously We have previously shown that C-domain+ exhibits activity 30-fold lower than WT (per monomer) for a protein substrate [14] Therefore, the PPIase activities of SIB1 FKBP22 and its derivatives for a protein substrate increase as fol-lows: C-domain+< NNC-FKBP22 < WT Likewise, the binding affinities of these proteins to a folding intermediate of protein increase in this order These results suggest that a monomeric form of FKBP22 with N- and C-domains is sufficient for PPIase activity for a protein substrate, but a V-shaped structure is required to increase it to the maximal activity
Stability of NNC-FKBP22 DSC analyses indicate that both domains of NNC-FKBP22 are more stable than the corresponding domains of WT by approximately 4C (Fig 3) The repetitive N-domains of NNC-FKBP22 are presumably folded into a structure similar to that of the N-domains of WT with a homodimeric structure This structure is more stable than that of WT, probably because a dimeric structure of the repetitive N-domains of NNC-FKBP22 is stabilized not only by hydrophobic interactions but also by covalent linkage through three glycine residues The covalent bond is known as the strongest chemical bond contributing to protein stability [37–40] In WT, a dimeric structure of the N-domains is stabilized only by noncovalent, mainly hydrophobic, interactions According to the crystal structures of L pneumophila MIP [11] and
E coli FkpA [12], the C-domain is completely sepa-rated from the N-domain Nevertheless, the C-domain
of NNC-FKBP22 is stabilized in parallel with its N-domain compared with the corresponding domains
of WT The C-domain is linked to the N-domain through the a3 helix Therefore, the C-domain of NNC-FKBP22 is probably indirectly stabilized when the N-domain is stabilized
It is noted that the optimum temperature for the activity of NNC-FKBP22 (10C) is identical to that
of WT, despite the fact that the catalytic domain of NNC-FKBP22 is more stable than that of WT by approximately 4 C According to the
C-domains start to unfold at temperatures that are considerably higher than the optimum temperatures for the activities of these proteins These results suggest that the local conformation around the active
Trang 8site is more sensitive to thermal denaturation than the
entire domain structure The stability of the local
conformation around the active site of NNC-FKBP22
may not be seriously changed compared with that of WT
Experimental procedures
Plasmid construction
Plasmid pSIB1-NNC, used to overproduce a His-tagged
form of NNC-FKBP22, was constructed using the PCR
overlap extension method [41] Plasmid pSIB1, used to
overproduce a His-tagged form of SIB1 FKBP22 [8], was
used as a template The sequences of the PCR primers used
are as follows: 5¢-AGAGAGAATTCATATGTCAGATT
TGTTCAG-3¢ for primer 1; 5¢-TTCCATACCACCACCT
GCAACTTGAAGCTC-3¢ for primer 2; 5¢-GTTGCAGGT
GGTGGTATGGAACAGCATGCT-3¢ for primer 3; and
5¢-GGCCACTGGATCCAACTACAGCAATTCTCA-3¢ for
primer 4 [the NdeI (primer 1) and BamHI (primer 4) sites
are underlined] Primers 1 (forward) and 2 (reverse) were
used to amplify the gene encoding Met1–Ala60 of SIB1
FKBP22, with three additional glycine residues at the
C-terminus Primers 3 (forward) and 4 (reverse) were used
to amplify the gene encoding Met8–Ile205, with three
addi-tional glycine residues at the N-terminus The resultant two
PCR fragments were combined and amplified by PCR using
primers 1 and 4 The PCR product was ligated into the
NdeI–BamHI sites of pET28a (Novagen, WI, USA) to
pro-duce pSIB1-NNC PCR was performed with the GeneAmp
PCR system 2400 (Applied Biosystems, Tokyo, Japan)
using KOD polymerase (Toyobo Co., Ltd., Kyoto, Japan)
The nucleotide sequence was confirmed using the Prism 310
DNA sequencer (Applied Biosystems) All oligonucleotides
were synthesized by Hokkaido System Science (Sapporo,
Japan)
Overproduction and purification
E coli BL21(DE3) [F) ompT hsdSB(rB)mB)) gal (kcI857
ind1 Sam7 nin5 lacUV5-T7gene1) dcm (DE3)] (Novagen)
was used as a host strain for the overproduction of
His-tagged SIB1 FKBP22 and NNC-FKBP22 Transformation
of the E coli cells with plasmid pSIB1 or pSIB1-NNC, and
overproduction and purification of the recombinant
proteins, were carried out as described previously for
His-tagged SIB1 FKBP22 [8] The production levels of the
recombinant proteins in the E coli cells, and their purities,
were analyzed by SDS–PAGE [42] using a 15%
polyacryl-amide gel, followed by staining with Coomassie Brilliant
Blue
Protein concentrations were determined from the UV
absorption on the basis that the absorbance at 280 nm of a
0.1% (1 mgÆml)1) solution is 0.68 for SIB1 FKBP22 and
0.61 for NNC-FKBP22 These values were calculated by using e = 1576 m)1Æcm)1 for Tyr and 5225 m)1Æcm)1 for Trp, at 280 nm [43]
Molecular mass Sedimentation equilibrium analytical ultracentrifugation was performed at 4C for 20 h with a Beckman Optima XL-A Analytical Ultracentrifuge (Beckman, Tokyo, Japan) using an An-60 Ti rotor at 140 000 g Before measurement, the protein solution was dialyzed overnight against 20 mm sodium phosphate (pH 8.0) at 4C The concentration of the protein for initial loading was 2 mgÆmL)1 Distribution
of the protein within the cell was analyzed by monitoring the absorbance at 280 nm Analysis of the sedimentation equilibrium was performed using the program xlavel, ver-sion 2 (Beckman)
Gel-filtration column chromatography was carried out using HPLC with a TSK-GEL G2000SWXL column
(Tos-oh Co., Tokyo, Japan) equilibrated with 50 mm Tris–HCl (pH 8.0) containing 50 mm NaCl Elution was performed
at a flow rate of 0.5 mLÆmin)1 Bovine tyroglobulin (670 kDa), bovine c-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.3 kDa) were used as standard proteins
Enzymatic activity The PPIase activity was determined using a protease-cou-pling assay [44] and an RNase T1 refolding assay [45] For the protease-coupling assay, chymotrypsin was used as a protease and Suc-ALPF-pNA (Wako Pure Chemical Indus-tries, Ltd., Osaka, Japan) was used as a substrate The reaction mixture (2 mL) contained 35 mm HEPES (pH 7.8), 25 lm Suc-ALPF-pNA and an appropriate amount of the enzyme The reaction mixture was incubated at the reaction temperature (4, 10, 15 or 20C) for 3 min before the addition of chymotrypsin The reaction was initiated by the addition of 30 lL of 0.76 mm chymotrypsin The isom-erization of the Leu–Pro bond, catalyzed by PPIase, was measured by monitoring the change in the concentration of p-nitroanilide (pNA), because pNA is released from the substrate only when this peptide bond is in a trans confor-mation The increase in the rate of isomerization is implicit
in the increased rate of pNA release, because catalysis of isomerization produces a trans substrate with increased fre-quency The concentration of pNA was determined from the absorption at 390 nm, with the molar absorption coeffi-cient value of 8900 M)1cm)1, using a Hitachi U-2010
UV⁄ VIS spectrophotometer (Hitachi High-Technologies Co., Tokyo, Japan) The catalytic efficiency (kcat⁄ Km) was calculated from the relationship kcat⁄ Km = (kp –kn)⁄ E, where E represents the concentration of the enzyme, and kp
and kn represent the first-order rate constants for the
Trang 9release of pNA from the substrate in the presence and
absence of the enzyme, respectively [46]
For the RNase T1 refolding assay, RNase T1 (16 lm)
(Funakoshi Co., Ltd., Tokyo, Japan) was first unfolded by
incubation in 20 mm sodium phosphate (pH 8.0),
contain-ing 0.1 mm EDTA and 6.2 m guanidine hydrochloride, at
10C overnight Refolding was then initiated by diluting
this solution 80-fold with 20 mm sodium phosphate (pH
8.0) containing 100 mm NaCl in the presence or absence of
the enzyme The final concentrations of RNase T1and the
enzyme were 0.2 lm and 10 nm, respectively The refolding
reaction was monitored by measuring the increase in
tryp-tophan fluorescence using an F-2000 spectrofluorometer
(Hitachi High-Technologies Co.) The excitation and
emis-sion wavelengths were 295 and 323 nm, respectively, and
the band width was 10 nm The refolding curves were
ana-lyzed with double exponential fit [36] The kcat⁄ Km values
were calculated from the relationship mentioned above,
where kpand kn represent first-order rate constants for the
faster refolding phase of RNase T1 in the presence and
absence of the enzyme, respectively
CD
The CD spectra were measured at 10C on a J-725
auto-matic spectropolarimeter (JASCO Co., Tokyo, Japan) The
protein was dissolved in 20 mm sodium phosphate (pH 8.0)
and incubated at 10C for 30 min before the measurement
was made For measurement of the far-UV CD spectra
(200–260 nm), the protein concentration was approximately
0.2 mgÆmL)1 and a cell with an optical path length of
2 mm was used For measurement of the near-UV CD
spectra (250–320 nm), the protein concentration was
approximately 0.7 mgÆmL)1and a cell with an optical path
length of 10 mm was used The mean residue ellipticity, h,
which has units of degÆcm2Ædmol)1, was calculated by using
an average amino acid relative molecular mass of 110
DSC
The DSC measurement was performed on a high-sensitivity
VP-DSC controlled by the VPVIEWERTM software
pack-age (Microcal Inc., Northampton, MA, USA) at a scan rate
of 1CÆmin)1 The protein was dissolved in 20 mm sodium
phosphate (pH 8.0) at approximately 1.0 mgÆmL)1 Before
performing the measurement, the protein solution was
fil-tered through 0.22-lm pore-size membranes and then
degassed in a vacuum The reversibility of thermal
denatur-ation was verified by reheating the sample
Surface plasmon resonance
The interaction between SIB1 FKBP22 or NNC-FKBP22
with reduced a-lactalbumins was monitored by surface
plas-mon resonance using the Biacore X instrument (Biacore, Uppsala, Sweden) Immobilization of the His-tagged pro-tein to a Ni2 +-chelated nitrilotriacetic acid sensor chip (Biacore) was carried out as described previously [15] Reduced a-lactalbumin, which was dissolved at a concen-tration of 100 lm in 20 mm sodium phosphate (pH 8.0) containing 2 mm dithiothreitol, 100 mm NaCl and 1 mm EDTA, was then injected at 10C, with a flow rate of
10 lLÆmin)1, onto the surface of the sensor chip on which the His-tagged protein was immobilized Binding surfaces were regenerated by washing with 0.5 m EDTA
To determine the dissociation constant, KD, the concen-tration of reduced a-lactalbumin injected onto the sensor chip was varied from 0.5 to 100 lm From the plot of the equilibrium-binding responses as a function of the concen-trations of reduced a-lactalbumin, the KD values were determined using steady-state affinity program of BIAevalu-ation Software (Biacore)
Acknowledgements
We thank Dr T Tadokoro for helpful discussions This work was supported, in part, by a grant (21380065) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Develop-ment Organization (NEDO) of Japan
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