We have previously shown that the cellular content of FKBP22 SIB1 FKBP22 from a psychrotrophic Keywords domain structure; FKBP22; PPIase; psychrotrophic bacterium; thermal stability Corr
Trang 1of FKBP22 from a psychrotrophic bacterium overproduced
in Escherichia coli
Yutaka Suzuki1, Kazufumi Takano1,2and Shigenori Kanaya1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
2 PRESTO, JST, Suita, Osaka, Japan
When polypeptides are synthesized at ribosomes,
pep-tide bonds are connected in trans form In the case of
peptide bonds N-terminal of the proline residues,
how-ever, some of them form cis peptide bonds in correctly
folded proteins [1] Consequently, trans-to-cis
conver-sions of these peptide bonds (prolyl isomerizations)
should occur during protein folding reactions As
dem-onstrated in some refolding experiments [2,3], prolyl
isomerizations are relatively slow and can be the rate
limiting step in protein folding reactions The cis-trans isomerizations of peptide bonds N-terminal of the pro-line residues are catalyzed by peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8) [4] Three structurally unrelated families of PPIases are known They are cyclophilins, parvulins, and FK506-binding proteins (FKBPs) [5]
We have previously shown that the cellular content
of FKBP22 (SIB1 FKBP22) from a psychrotrophic
Keywords
domain structure; FKBP22; PPIase;
psychrotrophic bacterium; thermal stability
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 27 September 2004, revised 23
October 2004, accepted 29 October 2004)
doi:10.1111/j.1742-4658.2004.04468.x
FKBP22 from a psychrotrophic bacterium Shewanella sp SIB1, is a
dimer-ic protein with peptidyl prolyl cis-trans isomerase (PPIase) activity Accord-ing to homology modelAccord-ing, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain
A long a3 helix spans these domains An N-domain with the entire a3 helix (N-domain+) and a C-domain with the entire a3 helix (C-domain+) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein C-domain+was shown to be a monomer and enzymatically active Its opti-mum temperature for activity (10C) was identical to that of the intact protein Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in sub-strate-binding The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5C and 46.6 C for the intact protein, and single heat absorption peaks at 44.7C for N-domain+ and 35.6C for C-domain+ These results indicate that the thermal unfolding transi-tions of the intact protein at lower and higher temperatures represent those
of C- and N-domains, respectively Because the unfolding temperature of C-domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibil-ity around the active site This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22
Abbreviations
ALPF, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; CD, circular dichroism; DSC, differential scanning calorimetry; FKBP, FK506-binding protein; MIP, macrophage infectivity potentiator; PPIase, peptidyl prolyl cis-trans isomerase.
Trang 2bacterium Shewanella sp SIB1 increases at 4C, as
compared to that at 20C [6] This protein is a member
of the macrophage infectivity potentiator (MIP)-like
FKBP subfamily proteins and shows amino acid
sequence identities of 56% to Escherichia coli FKBP22
[7], 43% to E coli FkpA [8], and 41% to Legionella
pneumophila MIP [9] SIB1 FKBP22 exists as a
homo-dimer and exhibits the PPIase activity like other
MIP-like FKBP subfamily proteins However, the optimum
temperature of this protein for activity (10C) is much
lower than that of E coli FKBP22 (> 25C) We
pose that this activity facilitates efficient folding of
pro-teins containing cis prolines in psychrotrophic bacteria
at low temperatures
According to the crystal structures of L pneumophila
MIP [10] and E coli FkpA [11], these proteins are
com-posed of N- and C-domains, which are spanned by a 40
amino acid long a3 helix The N-domain consists of a1
and a2 helices and an N-terminal region of a3 helix
The C-domain consists of six b-strands (b1–b6), a4
helix, and a C-terminal region of a3 helix The
N-domain is unique to the MIP-like FKBP subfamily
proteins This domain is involved in dimerization of the
protein and the interface between two monomers is
sta-bilized by the hydrophobic interactions of a1 and a2
helices In contrast, the C-domain (except a3 helix) is
conserved in all FKBP family proteins and contains the
entire PPIase active-site, suggesting that all FKBP
fam-ily proteins share a common catalytic mechanism The
a3 helix seems to be required to control the positions of
the two C-domains of the homodimer, such that these
domains are located with an appropriate distance and
orientation Because of the high similarity in the amino
acid sequence of SIB1 FKBP22 with that of L
pneumo-philaMIP and E coli FkpA, SIB1 FKBP22 might have
a similar three-dimentional structure
The stability–activity relationships of MIP-like FKBP
subfamily proteins remain to be analyzed Because the
prolyl isomerization is a spontaneous reaction and the
rate for this reaction increases as the reaction
tempera-ture increases, the PPIase activity cannot be accurately
determined at > 30C Therefore, it seems difficult to
analyze the stability–activity relationships of PPIases
from mesophilic and thermophilic organisms SIB1
FKBP22 seems to be an excellent model to analyze these
relationships because its optimum temperature for
activ-ity is 10C In addition, because this protein is expected
to consist of N- and C-domains, it would be informative
to construct the SIB1 FKBP22 variants containing
either one of these domains and compare their activities
and stabilities with those of the intact protein
In this report, the N- and C-domains of SIB1
FKBP22 were overproduced in E coli and purified in
an amount sufficient for physicochemical studies By comparing their activities and stabilities with those of the intact protein, we showed that the unfolding tem-perature of SIB1 FKBP22 is much higher than the optimum temperature for activity Based on these results, we discuss a role of each domain of SIB1 FKBP22 and a cold-adaptation mechanism of this protein
Results
Design
A model for the three-dimensional structure of the His-tagged form of SIB1 FKBP22 (SIB1 FKBP22*) was constructed based on the crystal structure of
L pneumophila MIP [10] (Fig 1) According to this model, SIB1 FKBP22 consists of N- and C-domains The N-domain of one molecule interacts with that of another molecule to form a homodimer The C-domain represents a catalytic domain Based on this model, three types of the SIB1 FKBP22 variants con-taining either one of these two domains were designed They are N-domain+, C-domain+, and C-domain– The primary structures of these variants are schemati-cally shown in comparison with that of the intact pro-tein in Fig 2 Because a long a3 helix spans both the N- and C-domains, and because the region containing only a1 and a2 helices seems to be too short to fold correctly, N-domain+ was designed such that
it contains the entire a3 helix Likewise, C-domain+ and C-domain– were designed such that the former
Fig 1 A tertiary model of SIB1 FKBP22 homodimer The a3 helix (Val52–Arg93), which spans both the N- and C-domains, is most deeply shaded The N-domain without a3 helix (Met1–Ala51), which
is involved in dimerization, is moderately shaded, and the C-domain without a3 helix (Asp94–Ile205), which is involved in catalytic func-tion, is most lightly shaded.
Trang 3contains the entire a3 helix and the latter does not
contain it
Overproduction and purification
Upon induction for overproduction at 10C,
N-domain+ and C-domain+ accumulated in the cells
in a soluble form, whereas C-domain– accumulated in
the cells in inclusion bodies (Fig 3) C-domain– was
solubilized in the presence of 6 m urea and refolded by
removing urea with a yield of nearly 100% All
pro-teins were purified to give a single band on
SDS⁄ PAGE (data not shown)
The molecular masses of N-domain+, C-domain+,
and C-domain– were estimated to be 15 kDa, 26 kDa,
and 17 kDa, respectively, by SDS⁄ PAGE (Fig 3)
These values are slightly larger than the calculated
ones from their amino acid sequences including a
His-tag (12 042 for N-domain+, 19 149 for C-domain+,
and 14 085 for C-domain–) The molecular mass of SIB1 FKBP22* estimated by SDS⁄ PAGE (29 kDa) has been reported to be larger than that determined by EMI-MS, which is identical to the calculated one (23 947) [6] Slow migration in the gel may be a char-acteristic common to SIB1 FKBP22* and its variants The molecular masses of N-domain+ and C-domain+ were also estimated to be 39 kDa and 23 kDa, respect-ively, by gel filtration column chromatography The former and latter values are larger than the calculated ones by 3.2 and 1.2 times, respectively, suggesting that N-domain+ exists as a trimer and C-domain+ exists
as a monomer However, the molecular mass of a dimeric form of SIB1 FKBP22* estimated by gel filtra-tion column chromatography has been reported to be larger than that determined by sedimentation equilib-rium analytical ultracentrifuge by 1.5 times [6] This discrepancy is probably caused by the unusual mole-cular shape of the protein, which is cylindrical rather
Fig 3 Estimation of the amount of the proteins in soluble and insoluble forms by SDS ⁄ PAGE N-domain + (A), C-domain – (B), and C-domain +
(C) were overproduced in E coli as described for SIB1 FKBP22* [6] The soluble (lane S) and insoluble (lane P) fractions after sonication lysis were analyzed by 15% (for C-domain + ) and 17% (for N-domain + and C-domain – ) SDS ⁄ PAGE The gel was stained with Coomassie Brilliant Blue Arrows indicate the recombinant proteins overproduced in the cells The positions of the standard proteins contained in a low mole-cular mass marker kit (Pharmacia Biotech, Piscataway, NJ, USA) are shown alongside the gels, together with their molemole-cular masses.
Fig 2 Schematic representations of the pri-mary structures of SIB1 FKBP22* and its variants A His-tag attached to the N-termini
of the proteins is represented by shaded box The a-helices and b-strands are repre-sented by cylinders and arrows, respect-ively These secondary structures are arranged based on a tertiary model of SIB1 FKBP22 Numbers indicate the positions of the residues relative to the initiator methio-nine residue The ranges of the N- and C-domains are also shown.
Trang 4than globular Because N-domain+ is expected to
assume a similar cylindrical structure, sedimentation
equilibrium analytical ultracentrifugation was
per-formed to determine its molecular mass in solution
The data fitted well to a single-species model with no
evidence of aggregation, and the molecular mass was
determined to be 23 431 Da This value is 1.9 times
larger than that calculated from the amino acid
sequence, indicating that N-domain+exists as a dimer
CD spectra
The far-UV CD spectra of N-domain+, C-domain+,
C-domain–, and SIB1 FKBP22* were measured at
10C (Fig 4A) The spectrum of N-domain+, which
gave a broad trough with a double minimum at 208
and 222 nm, was similar to that of SIB1 FKBP22*,
although the depth of the trough in this spectrum is
larger than that in the SIB1 FKBP22* spectrum
The helical content was calculated to be 51% for
N-domain+ and 38% for SIB1 FKBP22* from these
spectra using the method of Wu et al [12] These
val-ues were comparable to those calculated from a
ter-tiary model of SIB1 FKBP22* (60% for N-domain+
and 34% for SIB1 FKBP22*), suggesting that
N-domain+ assumes a similar helical structure to that
of the N-domain in the intact molecule On the other
hand, the CD spectra of C-domain+ and C-domain–
gave a broad trough with a single minimum at 207 nm
and one without any clear minimum, respectively The
depths of these troughs were considerably smaller than
that in the SIB1 FKBP22* spectrum
The near-UV CD spectra of these proteins were also
measured at 10C (Fig 4B) These spectra reveal the
three-dimensional environments of aromatic residues
such as Trp and Tyr SIB1 FKBP22* contains one
tryptophan residue (Trp157), which is conserved in the
FKBP family proteins and required for PPIase activity,
and seven tyrosine residues Because most of these
residues (one tryptophan and six tyrosine residues) are located in its C-domain, the near-UV CD spectrum of SIB1 FKBP22* may reflect the conformation of the C-domain The spectrum of C-domain+was similar to that of SIB1 FKBP22*, suggesting that C-domain+ assumes a similar structure to that of the C-domain
in the intact molecule In contrast, the spectrum of C-domain– was quite different from those of C-domain+ and SIB1 FKBP22*, suggesting that the structure of C-domain– is considerably different from that of the C-domain in the intact molecule
PPIase activity When the PPIase activity was determined at 10C
by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (ALPF) as a substrate, C-domain+ exhibited PPIase activity, whereas C-domain– did not The catalytic efficiency (kcat⁄ Km)
of C-domain+ was estimated to be 1.43 lm)1Æs)1, which was 1.6 times higher than that of SIB1 FKBP22* The temperature dependence of the PPIase activity of C-domain+ was nearly identical to that of SIB1 FKBP22* (Fig 5A) In contrast, when the PPI-ase activity was determined by the RNPPI-ase T1refolding assay, C-domain+exhibited much less activity as com-pared to that of SIB1 FKBP22* The acceleration effect of C-domain+ on the RNase T1 refolding reac-tion was not detected at 21 nm, but detected at 210 nm (Fig 5B) The acceleration effect similar to that detec-ted in the presence of 210 nm C-domain+was detected
in the presence of 19 nm SIB1 FKBP22* The kcat⁄ Km values were estimated to be 0.5 lm)1Æs)1 for SIB1 FKBP22* and 0.015 lm)1Æs)1for C-domain+
Thermal stability Heat induced unfolding of N-domain+, C-domain+, and SIB1 FKBP22* were analyzed by differential
Fig 4 CD spectra of SIB1 FKBP22* and its
variants The far-UV (A) and near-UV (B) CD
spectra of SIB1 FKBP22* (dashed line),
N-domain + (heavy thick line), C-domain – (thin
line), and C-domain + (moderately thick line)
are shown All spectra were measured at
10 C as described under Experimental
procedures.
Trang 5scanning calorimetry (DSC) (Fig 6, Table 1) All DSC
curves were reproduced by repeating thermal scans,
indicating that thermal unfoldings of these proteins are
highly reversible The denaturation curve of SIB1
FKBP22* clearly showed two well separated
transi-tions Deconvolution of the thermogram according to
a non-two-state denaturation model gives melting
tem-perature (Tm) values of 32.5C and 46.6 C for these
transitions These Tmvalues are nearly equal to those
of C-domain+ (35.6C) and N-domain+ (44.7C),
suggesting that the thermal unfolding transitions of
SIB1 FKBP22* at lower and higher temperatures
rep-resent those of its C-domain and N-domain,
respect-ively For unfolding of N-domain+, the van’t Hoff
enthalpy (DHvH) was roughly two times larger than the
calorimetric enthalpy (DHcal) Because N-domain+
exists as a dimer, this result possibly reflects a coupling
of the unfolding of N-domain+ to dissociation of the homodimer Similarly, the unfolding reaction of C-domain+ seems to contain complex processes, as indicated by theDHcal⁄ DHvHratio far from unity
Comparison of thermal stability of SIB1 FKBP22* and E coli FKBP22*
To examine whether SIB1 FKBP22* is less stable than its mesophilic counterpart, heat induced unfolding of
E coli FKBP22* was analyzed by DSC However, thermodynamic parameters including Tmcould not be obtained because of the poor reversibility of this pro-tein in thermal unfolding Therefore, thermal stabilities
of SIB1 FKBP22* and E coli FKBP22* were analyzed
by circular dichroism (CD) The far-UV CD spectra of SIB1 FKBP22* and E coli FKBP22* were measured
at various temperatures and the spectra of SIB1 FKBP22* at 10 and 50 C are shown in comparison with those of E coli FKBP22* at 20 and 80C in Fig 7 The spectrum of SIB1 FKBP22* at 10C is identical to that shown in Fig 4A The spectra of
Table 1 Thermodynamic parameters for heat induced unfolding of SIB1 FKBP22*, C-domain + and N-domain + recorded by microcalori-metry The melting temperature (Tm), calorimetric enthalpy (DH cal ), and van’t Hoff enthalpy (DH vH ) were obtained from the DSC curves shown in Fig 6, using ORIGIN software (MicroCal, Inc.).
Protein T m (C) DH cal (kJÆmol)1) DH vH (kJÆmol)1)
Fig 6 DSC curves of N-domain+, C-domain+, and SIB1 FKBP22*.
The DSC curves of N-domain + (thick line), C-domain + (thin line), and
SIB1 FKBP22* (dashed line), which were measured at a scan rate
of 1 CÆmin)1, are shown These proteins were dissolved in 20 m M
sodium phosphate (pH 8.0) at 0.6 mgÆmL)1.
Fig 5 PPIase activities of C-domain + (A) The temperature dependence of the PPIase activity of C-domain + (–d–), which was determined
by protease coupling assay using ALPF as a substrate, is shown in comparison with that of SIB1 FKBP22* (–s–) The catalytic efficiency,
kcat⁄ K m , was calculated according to Harrison & Stein [34] The experiment was carried out in duplicate Each plot represents the average value and errors from the average values are shown (B) The increase in tryptophan fluorescence at 323 nm during refolding of RNase T1 (0.2 l M ) is shown as a function of the refolding time Refolding reaction was carried out at 10 C in the absence (dotted line), or presence of
21 n M of C-domain + (thick solid line), 210 n M of C-domain + (thin solid line) or 19 n M of SIB1 FKBP22* (dashed line).
Trang 6SIB1 FKBP22* at 10C and E coli FKBP22* at
20C, which represent the spectra of these proteins in
a native form, were similar to each other, suggesting
that the tertiary structures of these proteins are similar
to each other With a temperature shift from 10 to
50C, the spectrum of SIB1 FKBP22*, which gave a
broad trough with double minimum [h] values of
)11 200 at 209 nm and )12 100 at 222 nm, was
greatly changed so that it exhibits a trough with a
minimum [h] value of )7800 at 207 nm, which is
accompanied by a shoulder with a [h] value of )5700
at 220 nm A similar spectral change was observed for
E coli FKBP22* when the temperature was shifted
from 20 to 80C The spectra of SIB1 FKBP22* at
50C and E coli FKBP22* at 80 C were not
seri-ously changed at higher temperatures, indicating that
these spectra represent the spectra of these proteins in
a denatured form In these conditions, SIB1 FKBP22*
was fully reversible in thermal denaturation, whereas
E coli FKBP22* was not The reversibility of E coli
FKBP22* was roughly 70%
The thermal denaturation curves of SIB1 FKBP22*
and E coli FKBP22* were measured by monitoring a
change in the CD values at 222 nm (Fig 8) SIB1
FKBP22* apparently unfolded through an
intermedi-ate stintermedi-ate The Tmvalues for the first and second
transi-tions were roughly estimated to be 32 and 44C,
respectively, which were comparable with those
determined by DSC As compared to SIB1 FKBP22*,
E coli FKBP22* unfolded at higher temperatures, indicating that it is more stable than SIB1 FKBP22* However, it is unclear whether this protein unfolds through an intermediate state as well, because this intermediate state was not clearly detected The ther-mal unfolding curve of this protein did not fit the the-oretical curve, which was drawn on the assumption that the protein unfolds in a single cooperative fashion (data not shown)
Discussion
Unfolding of SIB1 FKBP22*
In this study, SIB1 FKBP22* was shown to unfold in
a complex non-two-state mechanism with two peaks apparent in the DSC curve Construction of the N-domain+ and C-domain+, which lack the C- and N-domains, respectively, followed by DSC analyses, clearly showed that two peaks of heat capacity observed in thermal unfolding of SIB1 FKBP22* rep-resent unfoldings of its N- and C-domains In this thermal unfolding process, the C- and N-domains unfold at lower and higher temperatures, respectively
It has been reported that a phosphoglycerate kinase [13] and a chitobiase [14] from psychrophilic bacteria consist of a heat labile domain and a heat stable domain Bentahir et al [13] have proposed that a heat labile domain provides a sufficient flexibility around the active site, and a heat stable domain provides a sufficient rigidity to the substrate-binding site, so that
Fig 8 Thermal denaturation curves of SIB1 FKBP22* and E coli FKBP22* The [h] values of SIB1 FKBP22* (trace 1) and E coli FKBP22* (trace 2) at 222 nm are shown as a function of tempera-ture The proteins were dissolved in 20 m M sodium phosphate (pH 8.0) at 0.30 mgÆmL)1for SIB1 FKBP22* and 0.29 mgÆmL)1for
E coli FKBP22* A cell with an optical path length of 2 mm was used Temperature was linearly raised at 1 CÆmin)1.
Fig 7 Far-UV CD spectra of SIB1 FKBP22* and E coli FKBP22*.
The CD spectra of SIB1 FKBP22* measured at 10 C (thick line) and
50 C (thick dashed line), and those of E coli FKBP22* measured at
20 C (thin line) and 80 C (thin dashed line) are shown The spectra
were measured as described under Experimental procedures.
Trang 7the enzymatic reaction is efficiently achieved at low
temperatures Because the C-terminal catalytic domain
of SIB1 FKBP22 represents a heat labile domain, the
instability of this domain may be required to increase
the flexibility of the active-site at low temperatures
Stability and activity of SIB1 FKBP22*
SIB1 FKBP22* was shown to be much less stable than
E coli FKBP22* Its optimal temperature for activity
has been reported to be greatly shifted downward as
compared to that of E coli FKBP22* [6]
Cold-adap-tation has been specified by the increase in the
cata-lytic efficiency at low temperatures, the downward
shift in the optimum temperatures for activity, and the
reduction in the conformational stability [15]
There-fore, SIB1 FKBP22 can be defined as a cold-adapted
enzyme, although it is less active than E coli
FKBP22* even at low temperatures [6] Several
cold-adapted enzymes have also been reported to be less
active than their mesophilic counterparts [16–19]
Analyses of the thermal stability of SIB1 FKBP22*
by DSC (Fig 3) and CD (Fig 8) indicate that
unfold-ing of this protein is initiated at > 25C In fact, the
CD spectrum of SIB1 FKBP22* at 20C was nearly
identical to that at 10C (data not shown), suggesting
that the conformation of this protein is not seriously
changed upon temperature shift from 10 to 20C
Thermal unfolding of C-domain+ is also initiated
at > 25C Nevertheless, SIB1 FKBP22* and
C-domain+ both exhibit the maximal PPIase activity
at 10C and their activities are greatly reduced at
20C These results suggest that a subtle
conforma-tional change around the active-site causes a great
reduction of the enzymatic activity The large
differ-ence in the temperatures for enzymatic inactivation
and structural unfolding has been observed for
cold-adapted a-amylase and family 8 xylanase from an
Antarctic bacterium [20,21] The apparent optimal
temperatures of these proteins for enzymatic activities
are much lower than the temperatures at which any
significant conformational event occurs In contrast,
the optimal temperatures for the activities of their
mesophilic and thermophilic counterparts closely
cor-relate with the temperatures for their structural
transi-tions Thus, the large difference in the temperatures
for enzymatic inactivation and structural unfolding
seems to be a characteristic feature of cold-adapted
enzymes It has been proposed that this difference is
caused by a cold-adaptation strategy termed ‘localized
flexibility’ [20] Although an increase in flexibility
around the active site increases kcat by reducing the
energy cost of conformational change during the
cata-lytic reaction, it should increase Km concomitantly By restricting the increase of flexibility within small areas, cold-adapted enzymes prevent unfavorable increases in
Km [22] SIB1 FKBP22 probably adopts a similar strategy for cold-adaptation
Structural importance of a3 helix
Two types of the SIB1 FKBP22* variants, which con-tain the C-domain, were designed based on its tertiary model C-domain+ contains an entire a3 helix, whereas C-domain– does not contain it These two proteins differ greatly in their biochemical properties C-domain+ was overproduced in E coli in a soluble form and exhibited the PPIase activity Its near-UV
CD spectrum was similar to that of SIB1 FKBP22*
In contrast, C-domain– was overproduced in E coli in inclusion bodies and exhibited little PPIase activity Its near-UV CD spectrum was quite different from that of SIB1 FKBP22* These results strongly suggest that a3 helix is required to facilitate folding of the C-domain,
or to stabilize it, so that the C-domain assumes a native conformation It has previously been reported that limited proteolysis of L pneumophila MIP allows the separation of their N- and C-domains such that the C-domain contains the C-terminal half of the a3 helix [23,24] In addition, the C-domain of E coli FkpA shows a high tendency to form inclusion bodies when it is overproduced in E coli in a form without a3 helix [25] These results are consistent with our results According to the crystal structure of L pneu-mophila MIP, there are three distinct contacts between the C-terminal region of a3 helix and the C-domain [10] These contacts may also be conserved in the structure of SIB1 FKBP22
Role of N- and C-domains Most organisms contain multiple PPIases within a sin-gle cell They are usually composed of several domains; one is common to the members of each family and specifies the family to which that PPIase belongs, and the others are unique to the particular PPIase and thought to be related to the protein’s distinct function The C- and N-domains of MIP-like FKBP subfamily proteins represent the former and latter domains, respectively Therefore, biochemical charac-terizations of N-domain+ and C-domain+ will facili-tate understanding of the roles of these domains in the intact molecule
The observation that N-domain+ exists as a dimer, whereas C-domain+ exists as a monomer supports a tertiary model of SIB1 FKBP22, in which the a1 and a2
Trang 8helices form the dimerization core of the protein In
addition, we showed that the PPIase activity of
C-domain+determined by the RNase T1refolding assay
was greatly reduced as compared to that of the intact
protein These results suggest that a dimeric structure of
SIB1 FKBP22 is responsible for its high PPIase activity
for protein substrates Alternatively, N-domain contains
a binding site for protein substrates Similar results have
been reported for other MIP-like FKBP subfamily
pro-teins For example, The C-domain of L pneumophila
MIP produced upon limited proteolysis has been
repor-ted to exist as a monomer and exhibit weak PPIase
activity for protein substrate [23] Likewise, the
C-domain of E coli FkpA is devoid of chaperone-like
function, although it shows PPIase activity [11,25]
Fur-thermore, it has been reported that human FKBP12
which intrinsically consists of a single domain, exhibited
lower activity for RNase T1substrate and higher activity
for tetrapeptide substrates than E coli FkpA [25]
How-ever, the reason why C-domain alone exhibits a weak
activity for protein substrates remains to be clarified
Further structural and functional studies of these
pro-teins will be required to clarify this reason
Experimental procedures
Cells and plasmids
Psychrotrophic bacterium Shewanella sp SIB1 was isolated
from water deposits in a Japanese oil reservoir [26] E coli
JM109 [recA1, supE44, endA1, hsdR17, gyrA96, relA1, thi,
D(lac-proAB) ⁄ F¢, traD36, proAB+, lacIq lacZDM15] was
obtained from Toyobo Co., Ltd (Kyoto, Japan) E coli
BL21(DE3) [F–, ompT, hsdSB (rB–, mB–), gal, dcm (DE3)]
and plasmid pET-28a were obtained from Novagen
(Madi-son, WI, USA) Plasmid pUC18 was obtained from Takara
Shuzo Co., Ltd (Kyoto, Japan) The E coli transformants
were grown in Luria–Bertani medium containing 50 mgÆL)1
ampicillin or 35 mgÆL)1kanamycin
Plasmid construction
Plasmid pSIB1-Nd, pSIB1-Cd, and pSIB1-a3+Cd for
over-production of a His-tagged form of the N-domain of SIB1
FKBP22 with entire a3 helix (N-domain+), C-domain
with-out a3 helix (C-domain–), and C-domain with entire a3 helix
(C-domain+), respectively, were constructed by ligating a
part of the SIB1 FKBP22 gene amplified by PCR into
pET-28a as follows Genomic DNA was prepared from a Sarkosyl
lysate of the Shewanella sp SIB1 cells [27] and used as a
tem-plate The gene encoding Met1–Asp94 of SIB1 FKBP22 was
amplified by PCR and ligated into the NdeI–SacI sites of
pET-28a to produce plasmid pSIB1-Nd Likewise, the genes
encoding Gly95–Ile205 and Gly47–Ile205 of SIB1 FKBP22 were amplified by PCR and ligated into pET-28a to produce plasmids pSIB1-Cd and pSIB1-a3+Cd, respectively The sequences of the 5¢ PCR primers were 5¢-AGAGAGAA TTCATATGTCAGATTTGTTCAG-3¢ for N-domain+,
AGAAAGC-3¢ for C-domain+
, where underlined bases show the position of the NdeI site The sequences of the 3¢ PCR primers were 5¢-GACTCTGAGCTCGTAATCTAGT CACGCTTA-3¢ for N-domain+, where underlined bases show the position of the SacI site, and 5¢- GGCCACT
and C-domain+, where underlined bases show the position
of the BamHI site PCR was performed with GeneAmp PCR system 2400 (PerkinElmer, Tokyo, Japan) using KOD polymerase (Toyobo Co., Ltd) according to the procedures recommended by the supplier
Overproduction and purification His-tagged forms of SIB1 FKBP22 (SIB1 FKBP22*) and
E coli FKBP22 (E coli FKBP22*) were overproduced and purified as described previously [6] N-domain+, C-domain–, and C-domain+ were overproduced in the
E coli BL21(DE3) cells transformed with plasmids
pSIB1-Nd, pSIB1-Cd, and pSIB1-a3+Cd, respectively, and puri-fied, as described for SIB1 FKBP22* [6], except for the purification of C-domain– For purification of C-domain–, which was overproduced in inclusion bodies, the cells were disrupted by sonication and centrifuged at 15 000 g for
30 min at 4C The pellet was dissolved in 20 mm sodium phosphate (pH 8.0) containing 6 m urea and 0.5% (w⁄ v) Triton X-100, and incubated overnight at 4C After cen-trifugation at 15 000 g for 30 min at 4C to remove insol-uble materials, the protein was refolded by dialysis against
20 mm sodium phosphate (pH 8.0), and purified as des-cribed for SIB1 FKBP22* using metal chelating affinity chromatography and gel filtration chromatography [6] Production of the recombinant proteins in the E coli cells, as well as their purities, were analyzed by SDS⁄ PAGE [28] on a 15 or 17% polyacrylamide gel, followed by stain-ing with Coomassie Brilliant Blue
Protein concentration Protein concentrations were determined from the UV absorption on the basis that the absorbance at 280 nm of
a 0.1% solution is 0.68 for SIB1 FKBP22*, 0.12 for N-domain+, 1.01 for C-domain–, 0.75 for C-domain+and 0.69 for E coli FKBP22* These values were calculated by
using ¼ 1576 m)1Æcm)1 for Tyr and 5225 m)1Æcm)1 for Trp at 280 nm [29] For N-domain+, which contains only one tyrosine residue and no tryptophan residues, a method
Trang 9of Scopes [30] was used to confirm the accuracy of its
concentration In this method, the protein concentration
(mgÆmL)1) is calculated from A205nm⁄ (31 · b), where
A205nm represents absorbance at 205 nm and b represents
an optical path length (cm)
Molecular mass
The molecular masses of purified proteins were estimated by
gel filtration column chromatography using a Superdex 200
16⁄ 60 gel filtration column (Amersham Biosciences,
Piscat-away, NJ, USA) 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 serum albumin (67 kDa),
ovalbumin (44 kDa), chymotrypsinogen A (25 kDa), and
RNase A (14 kDa) were used as standard proteins
The molecular mass of N-domain+in solution was
deter-mined by sedimentation equilibrium analytical
ultracentri-fugation Sedimentation equilibrium experiments were
performed at 10C for 20 h with a Beckman Optima XL-A
Analytical Ultracentrifugate using an An-60 Ti rotor at a
speed of 28 000 r.p.m Before measurements, the protein
solutions were dialyzed overnight against 20 mm sodium
phosphate (pH 8.0) at 4C The initial loading
concentra-tion of the protein was 1.8 mgÆmL)1 The protein
concen-tration distribution within the cell was monitored by the
absorbance at 280 nm Analysis of the sedimentation
equili-bria was performed using the program xlavel (Beckman,
Tokyo, Japan, version 2)
Enzymatic activity
The PPIase activity was determined by protease-coupling
assay [31,32] and RNase T1 refolding assay [33] For the
protease-coupling assay, chymotrypsin was used as the
protease and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide
(ALPF; Wako Chemicals, Osaka, Japan) was used as the
substrate The reaction mixture (2.1 mL) contained 35 mm
Hepes buffer (pH 7.8), 25 lm tetrapeptide substrate, and
the appropriate amount of the enzyme The reaction
mix-ture was incubated at reaction temperamix-ture (4, 10, 15 or
25C) for 3 min prior to the addition of chymotrypsin
The reaction was initiated by the addition of 30 lL of
0.76 mm chymotrypsin The isomerization reaction
cata-lyzed by PPIases was measured by monitoring the change
in the concentration of p-nitroaniline, because p-nitroaniline
is not released from the substrate when the peptide bond
N-terminal of the proline residue is in the cis conformation
The concentration of p-nitroaniline was determined from
the absorption at 390 nm with the molar absorption
coeffi-cient value of 8900 m)1Æcm)1 using a Hitachi U-2010
UV⁄ VIS spectrophotometer (Hitachi Instruments, Tokyo,
Japan) The catalytic efficiency (kcat⁄ Km) was calculated
from the relationship kcat⁄ Km¼ (kp– kn)⁄ E, where E
repre-sents the concentration of the enzyme, and kp and kn
represent the first-order rate constants for the release of p-nitroaniline from the substrate in the presence and absence of the enzyme, respectively [34]
For the RNase T1 refolding assay, RNase T1 was first unfolded by incubating the solution containing 50 mm Tris⁄ HCl (pH 8.0), 1 mm EDTA, 5.6 m guanidine hydro-chloride, and 16 lm RNase T1(Funakoshi, Tokyo, Japan) at
10C overnight Refolding was then initiated by diluting this solution 80-fold with 50 mm Tris⁄ HCl (pH 8.0) containing SIB1 FKBP22* or C-domian+ The final concentrations of RNase T1, SIB1 FKBP22*, and C-domian+ were 0.2 lm,
19 nm, and 21 or 210 nm, respectively The refolding reaction was monitored by measuring the increase in tryptophan fluorescence with an F-2000 spectrofluorometer (Hitachi Instruments) The excitation and emission wavelengths were
295 and 323 nm, respectively, and the band width was
10 nm The refolding curves were analyzed with double expo-nential fit [35] The kcat⁄ Kmvalues were calculated from the relationship described above, where kpand knrepresent the first-order rate constants for the faster refolding phase of RNase T1 in the presence and absence of the enzyme, respectively
Circular dichroism The CD spectra were recorded on a J-725 automatic spec-tropolarimeter from Japan Spectroscopic Co., Ltd (Tokyo, Japan) The proteins were dissolved in 20 mm sodium phos-phate (pH 8.0) and incubated for 30 min at the temperatures indicated prior to the CD measurement For measurement
of the far-UV CD spectra (200–260 nm), the protein concen-tration 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 (240–320 nm), the protein concen-tration was 0.4–1.0 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 molecular mass of 110
Differential scanning calorimetry DSC measurements were carried out on a high-sensitivity VP-DSC controlled by the vpviewerTM software package (Microcal, Inc., Northampton, MA, USA) at a scan rate of
1CÆmin)1 Prior to the measurements, samples were fil-tered through 0.22 lm pore size membranes and then de-gassed in a vacuum The protein concentrations during the measurements were 0.5 mgÆmL)1 The reversibility of thermal denaturation was verified by reheating the samples
Homology modeling
A model for dimeric structure of SIB1 FKBP22 was built
by SWISS-MODEL (Swiss Institute of Bioinfomatics)
Trang 10[36,37] using the structure of L pneumophila MIP (PDB
ID: 1fd9) as a template
Acknowledgements
We thank K Ogasahara (Institute for Protein Research,
Osaka University) for use of Hitachi U-2010 UV⁄ VIS
spectrophotometer and microcal DSC, and Dr M
Morikawa for helpful discussions This work was
sup-ported in part by a Grant-in-Aid for National Project
on Protein Structure and Functional Analyses and by a
Grant-in-Aid for Scientific Research (No 16041229)
from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and by a research
grant from the Noda Institute for Scientific Research
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