Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 489-496
doi:10.1111/j.1745-7270.2008.00426.x
Expression and purification of cysteine
mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear
magnetic resonance study
Jiafu Liu1, Kejiang Lin1, Chenyun
Guo2, Hongchang Gao2, Yihe Yao2, and Donghai Lin1,2*
1
School of Life Science
and Technology, China Pharmaceutical University, Nanjing 210009, China
2
Shanghai Institute of
Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 201203, China
Received: March 3,
2008 Accepted: April 30,
2008
This work was
supported by the grants from the National Natural Science Foundation of China
(30730026 and 30570352)
*Corresponding
author: Tel, 86-21-58433609; Fax, 86-21-50806036; E-mail,
Lipocalin-type prostaglandin (PG) D
synthase (L-PGDS) is the only member of the lipocalin superfamily that displays
enzymatic activity. It binds lipophilic ligands with high affinity and also can
catalyze PGH2 to produce
PGD2. Three cysteine residues, Cys65, Cys89, and Cys186 in L-PGDS, are conserved among all species, of which
Cys89 and Cys186 residues form a disulfide bridge. In this study, we
clarified the effects of thiol groups on the structure of the protein and
investigated the structural significance of Cys residues of rat L-PGDS by
site-directed mutagenesis. Four mutants were constructed by substituting Cys
residues with alanine to identify the correct formation of disulfide bonds among
these three residues. The effects of thiol groups on the structure of rat
L-PGDS were also identified by these mutants. Analysis of HSQC experiments
indicated that these enzymes were all properly folded with well defined
tertiary structures. As the first step towards the 3-D nuclear magnetic
resonance solution structure, we optimized expression of recombinant rat L-PGDS
in Escherichia coli and established an efficient and economic
purification protocol yielding large amounts of pure isotopically labeled rat
L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS
obtained using this expression system was suitable for determination of 3-D
nuclear magnetic resonance solution structure.
Keywords lipocalin-type prostaglandin D synthase;
mutant; HSQC; heteronuclear NMR
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) has been known
and studied for many years under another name, b-trace. It is abundantly
expressed in the central nervous system of various mammals, male genitals, and
human heart [1]. In these tissues, L-PGDS catalyzes the isomerization of a
common precursor of various prostanoids, PGH2, to
produce PGD2, an endogenous somnogen [2] and a modulator of pain responses [3],
in the presence of sulfhydryl compounds [4]. L-PGDS is the only enzyme among
members of the lipocalin superfamily [5], which includes a group of
lipid-transporter proteins such as epididymal retinoic acid-binding protein
[6], retinol-binding protein, b-lactoglobulin, major urinary protein, and tear lipocalin [7].
L-PGDS also acts as a lipophilic ligand-binding protein and it binds
biliverdin, bilirubin, retinaldehyde, and retinoic acid with high affinities in
vitro [8]. Thus, this protein is considered to be a dual functional
protein.The xenogenous L-PGDS proteins share a number of common sequences
and structural features. They all belong to N-glycosylated monomeric protein
with 180–190 amino acid residues and a molecular mass of 20–31 kDa,
depending on the size of the glycosyl moiety [9]. A signal peptide of
approximately 20 amino acid residues is commonly observed at the N-terminus of
L-PGDS and is removed to form the mature enzyme. Cysteine residues Cys89 and Cys186 of L-PGDS form a disulfide bridge that is
highly conserved among most, but not all, lipocalins [5]. The recent study into
the nuclear magnetic resonance (NMR) solution structure of mouse L-PGDS
showed that L-PGDS shares the common structural feature of the lipocalin
superfamily [10], the presence of a six- or eight-stranded b-barrel in their
tertiary structure [11]. Previous studies showed that thiol groups have
important effects on biological and structural characteristics. Furthermore,
proteins often undergo conformational changes to execute their biological
functions, such as an enzyme reaction or ligand binding [12], and structural
stability of the protein is closely related to its biological function [13].
Investigation of structural characteristics of one protein can not only provide
new information concerning the reaction mechanism of the enzyme and ligand
binding, but also help address many questions on the physiological functions
[14]. Thus, it is necessary to clarify the effects of thiol groups on the
structure of the protein that we are investigating.In the present study, we clarified the effects of thiol groups on
the structure of the protein and investigated the structural significance of
Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were
constructed by the replacement of cysteine (Cys) with alanine (Ala), namely C186A, C65A, C89,186A, and C65,89,186A mutants. The C65,89,186A mutant was used as a
reference during some experimental processes. These four mutants could help us
to identify the correct disulfide pairing during the expression and
purification processes. Among these isoforms, we selected a suitable protein
for further NMR studies. To further determine the NMR solution structure of rat
L-PGDS, we optimized the expression in Escherichia coli and established
an economic purification protocol yielding sufficient amounts of properly
folded and highly pure isotopically labeled enzyme.
Materials and Methods
Site-directed mutagenesis and plasmid construction
The rat L-PGDS gene in pGEM-T Easy vector, previously
provided by Prof. Yonglian Zhang (Institute of Biochemistry and Cell Biology,
Shanghai Institutes of Biological Science, Chinese Academy of Sciences,
Shanghai, China), was used as a template to clone wild-type protein and
mutants. All of the recombinant proteins lack the 24 amino acid long N-terminal
signal peptide. The GB1-fusion expression vector, pGBTNH, was used in this
study, which was constructed based on the pET-22b vector and GB1 as a partner
protein [15]. The coding sequences of L-PGDS isoforms were amplified by
polymerase chain reaction using appropriate primers, then were inserted into
bacterial expression vector pGBTNH at the cloning sites of BamHI and XhoI.
The successful cloning was confirmed by DNA sequencing. In this study, we
constructed four mutants by the replacement of Cys with Ala, namely C186A, C65A, C89,186A, and C65,89,186A mutants.
Expression and purification of proteins
The expression vectors harboring each enzyme gene were transformed
into E. coli BL21(DE3). Expression and purification of the fusion
proteins were carried out using the following procedures. Ten milliliters of
the overnight culture was added into 1000 ml of LB media and shaken at 37 ?C.
When the OD600 of the culture reached 0.7, 0.25 mM IPTG was added to induce
protein expression with shaking at 37 ?C for 4 h. The cells were harvested by
centrifugation (5000 g, 10 min, 4 ?C), resuspended in buffer A [50 mM
phosphate, 500 mM NaCl (pH 8.0), and 0.5 mM phenylmethylsulphonyl fluoride],
lysed by sonication, and centrifuged (11,000 g, 45 min, 4 ?C). The
supernatant was then loaded onto an Ni-NTA affinity column (Qiagen, Hilden,
Germany) previously equilibrated with buffer A. After the column was washed
using a gradually increasing concentration of imidazole, the GB1-fusion protein
was eluted with 250 mM imidazole in buffer A. The supernatant containing the
GB1-fusion proteins was further purified and desalted by a fast performance
liquid chromatography (FPLC) system equipped with a Superdex 75 10/300 GL
column (GE Healthcare, Wisconsin, USA), previously equilibrated then washed
with buffer B [15 mM phosphate and 150 mM NaCl (pH 6.5)]. Then the GB1-tag was
removed from GB1-fusion protein with bovine thrombin (Sigma, St. Louis, USA)
in buffer B [5 U thrombin/(mg fusion protein), incubation for 12 h at 25 ?C].
After thrombin cleavage, the fractions were loaded onto an Ni-NTA affinity
column and an FPLC system again, as above, to obtain pure samples. The purified
fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (15%) and non-denatured gel electrophoresis followed
by Coomassie staining. The solution samples were stored at room temperature and
the stabilities of proteins were monitored once a week by SDS-PAGE
electrophoresis for 1 month.
Preparation of isotopically labeled samples
Isotopically labeled samples were prepared according to the method
of Marley et al [16]. Uniformly 15N-labeled
samples for 1H
–15N HSQC spectra were prepared in
500 ml minimal M9 media containing 2 mM MgSO4, 0.1 mM
CaCl2, 15.1 g/L Na2HPO4·12H2O, 3 g/L KH2PO4, 0.5
g/L NaCl, 3 mg/L FeCl3, 100 mg/ml ampicillin, 2 g/L D-glucose,
and 1 g/L 15NH4Cl (Cambridge Isotope Laboratories, Andover,
USA) as the only source of nitrogen. When the OD600 of
the culture reached 0.4–0.6 after inoculation of recombinant E. coli BL21, induction
was carried out by adding 0.25 mM IPTG. Cells were harvested after 5–6 h of
expression and target proteins were purified as described above.
For the preparation of 13C/15N-labeled samples used to record 3-D heteronuclear NMR spectra, 13C-glucose (Cambridge Isotope Laboratories) was used as the only
source of carbon in M9 media. All other steps are similar to those for the
preparation of 15N-labeled samples.
Circular dichroism (CD) spectroscopy
CD spectra were recorded on a J-810 spectropolarimeter (Jasco,
Tokyo, Japan) at room temperature. The protein solutions contained rat L-PGDS
(200 mg/ml) in buffer B. The protein concentration was determined by the
absorption at 280 nm using calculated extinction coefficients. The parameters
for far-ultraviolet (UV) CD measurements were: a cylindrical cell of 0.1 cm
path length; 1 nm bandwidth; 0.1 nm resolution; 0.25 s response time; and a
scan speed of 100 nm/min. Each spectrum was the accumulation of three scans
from 190 to 250 nm followed by noise reduction.CD spectra were recorded on a J-810 spectropolarimeter (Jasco,
Tokyo, Japan) at room temperature. The protein solutions contained rat L-PGDS
(200 mg/ml) in buffer B. The protein concentration was determined by the
absorption at 280 nm using calculated extinction coefficients. The parameters
for far-ultraviolet (UV) CD measurements were: a cylindrical cell of 0.1 cm
path length; 1 nm bandwidth; 0.1 nm resolution; 0.25 s response time; and a
scan speed of 100 nm/min. Each spectrum was the accumulation of three scans
from 190 to 250 nm followed by noise reduction.
Electrospray ionization mass spectrometry (ESI-MS)
Mass spectra of these enzymes were recorded on a Micromass LCT
electrospray time-of-flight mass spectrometer (Waters, Milford, USA) with an
electrospray interface and in positive ion mode. The spray tip potential was
2000 V, the sample cone potential was 50 V, and the desolvation temperature was
150 ?C. As rat L-PGDS and its mutants are highly soluble, we dissolved
lyophilized samples in deionized water at 20 mM. The data were recorded
and processed using Masslynx 3.5 software (Waters).
1H-15N HSQC NMR spectroscopy
Uniformly 15N-labeled samples for NMR spectroscopy were
dissolved in NMR buffer [15 mM sodium phosphate, and 150 mM NaCl (pH 6.5)]
containing 10% D2O, and the protein concentrations were set at
1 mM in 250 ml in a 5 mm microcell NMR tube (Shigemi, Tokyo, Japan). 1H-15N HSQC spectra were measured with a Unity
Inova-600 spectrometer (Varian, Palo Alto, USA) equipped with a triple
resonance probe. Water suppression was achieved by solvent presaturation. The
temperature for NMR experiments was set at 25 ?C. Phase-shifted sine-bell
window functions, solvent-suppression filter on the time-domain data, and
zero-filling were applied before Fourier transformation.
3-D heteronuclear NMR experiments
All of the following NMR experiments were carried out on a Varian
Unity Inova-600 NMR spectrometer. Proteins in NMR buffer were concentrated to 1
mM by a Centricon filter unit (Millipore, Billerica, USA), then 10% D2O was added for the field-lock, and a suite of 3-D heteronuclear NMR
experiments was obtained. The following 3-D triple-resonance experiments were
conducted to obtain sequence-specific assignments for the backbone of rat
L-PGDS: HNCACB, CBCACONH, HNCO, HN(CA)CO, HNCA, HN(CO)CA, CBCA(CO)NH, and
HBHANH. The NMR experiments of C(CO)NH, H(CCO)NH, CCH-TOCSY, HCCH-TOCSY, and 15N-edited TOCSY-HSQC were carried out to obtain side-chain assignment.
The NMR spectra of 13C-edited NOESY-HSQC and 15N-edited NOESY-HSQC were collected to obtain interproton distance
restraints. All spectra were processed by NMRPipe and NMRDraw software [17] and
analyzed using the Sparky 3 program [18]. All software was run on a Linux
system. Linear prediction and zero-filling approaches were used to obtain
complex data matrices of 1024(t3)?256(t1)?128(t2)
before Fourier transformation to improve resolution. The water flip-back
approach was used in HSQC-type experiments to minimize saturation of water
resonance.
Results
Bacterial production of rat L-PGDS protein
Rat L-PGDS and its mutants were expressed in E. coli strain
BL21(DE3). The pGBTNH vector was used to obtain the soluble proteins for NMR
studies. In this system, wild-type enzyme and four mutants were all
produced in soluble state. The C-terminal His-tag allowed purification of the
proteins using chromatography on an Ni-NTA column. After incubation with
thrombin, rat L-PGDS and mutants were further purified by an FPLC system on
Superdex 75 to apparent homogeneity to be more than 95% pure as judged by
SDS-PAGE [Fig. 1(A)]. Under reducing conditions, the wild-type enzyme
and four mutants gave a single band at the same position with a molecular mass
of 19 kDa. The yield of enzymes per liter of bacterial culture varied between
50 mg
and 70 mg, and the yields of C186A and C89,186A mutants were apparently higher than that of C65,89,186A, C65A, and wild-type enzymes. On non-denatured gel
electrophoresis, all enzymes migrated as a homogenous band, suggesting that
they are monomeric proteins [Fig. 1(B)]. They were stable on storage in
a refrigerator for seven weeks or at room temperature for one month. They were
highly soluble and could be concentrated to 30 mg/ml without any visible
precipitation.
Secondary structure analysis
To confirm the proper folding of the bacterially produced rat L-PGDS
and its mutants, the proteins were analyzed by CD spectroscopy. The wavelength
range of CD (190–250 nm) is mainly determined by the main-chain conformation and,
therefore, far-UV CD spectra provide information about the secondary structure
of a protein [19]. In this study, the far-UV CD spectra of rat L-PGDS and
mutants showed general similarity (Fig. 2), revealing the typical features
of a predominant b-sheet protein [20] with a negative maximum between 205 and 219 nm
and a positive band below 200 nm. The far-UV CD spectra further showed three
resolved negative peaks at approximately 208, 219, and 222 nm, suggesting that
they all belong to the a+b class of proteins [21]. All enzymes have similar CD spectra
composed of 18% a-helix, 46% b-sheet, and 36% coil, indicating that they have a similar secondary
structure and all fold properly.
Characterization of rat L-PGDS enzymes by ESI-MS
MS was used to further check the purity and identity of these
enzymes. The ESI-MS spectra of wild-type rat L-PGDS and its mutants indicated
that they were all highly pure. The ESI-MS spectra of mutants were similar to
that of wild-type rat L-PGDS. The molecular mass of wild-type rat L-PGDS was
determined to be 20020.1 Da (Fig. 3). This value was in agreement with
the theoretical molecular mass, 20017.4 Da, which is calculated from the amino
acid sequence, including two additional residues (GS) at the N-terminus and
eight residues (LEHHHHHH) at the C-terminus that were included in the expressed
protein for cloning and purification purposes. Molecular masses of C186A, C65A, and C89,186A
mutants were measured to be 19984.2, 19987.2, and 19956.0 Da, respectively
(ESI-MS of these three mutants not shown). Their theoretical molecular masses
were 19985.3, 19985.3 and 19953.3 Da, respectively.
Structural changes monitored by 2-D HSQC experiments
The 2-D 1H-15N HSQC experiment
involving more than one type of nucleus is referred to as a heteronuclear
experiment and is often used in protein studies. It is an 1H-15N correlation experiment that generates cross
peaks resulting from amide proton and amide nitrogen. Each amide group,
including that of side-chains, in amino acid residues (except proline) gives
one single resonance peak in the 2-D 1H-15N HSQC
spectrum. Rat L-PGDS without signal peptide consists of 165 amino acids
including eight prolines that do not contribute signals to the 1H-15N HSQC spectrum. Consequently, together with
the side-chain amide protons, two additional residues (GS) at the N-terminus,
and eight residues (LEHHHHHH) at the C-terminus, approximately 180 cross peaks
would be expected to show in the spectrum. The high dispersion of signals in both
dimensions indicates the proper folding and the well defined tertiary
structures of rat L-PGDS and its mutants (Fig. 4). The effects of disulfide on the protein structure were obtained by
comparing HSQC spectra of proteins with and without disulfide bonds. Overlayed 1H-15N HSQC spectra of rat L-PGDS with disulfide
bonds and C186A mutant without disulfide bonds indicated that they did not adopt
absolutely the same structure [Fig. 4(A)]. However, the relative sites
of two identical amino acids from two isoforms in 1H-15N HSQC spectrum indicated that they adopted a similar conformation;
this was also consistent with the results of CD experiments. Overlayed 1H-15N HSQC spectra of wild-type L-PGDS and C65A mutant indicated that they adopted the same backbone structure [Fig.
4(B)]. This further confirmed that Cys89 and Cys186 residues formed a disulfide bridge. Overlayed 1H-15N HSQC spectra of C89,186A
mutant and C186A mutant, both without disulfide bonds, also indicated that they adopted
the same backbone structure [Fig. 4(C)]. On the basis of small
distinctions on structures of rat L-PGDS and its isoforms, we selected the
wild-type rat L-PGDS, which was closest to the native state, for the
determination of the 3-D solution structure by NMR spectroscopy.
Assignments of wild-type rat L-PGDS
To obtain the 3-D solution structure of proteins and to prepare the
following NMR studies, we first assigned the resonance peaks in NMR spectra using
Sparky software. We usually classify the amino acid spin systems or amino acid
residue types and establish the heteronuclear scalar coupling in triple
resonance experiments such as HNCA, HN(CO)CA, HN(CO)CACB, and HNCACB. Then we
establish sequential connectivities between these spin systems using
through-space coupling in NOESY experiments such as 13C-edited
NOESY-HSQC and 15N-edited NOESY-HSQC [22]. At
the beginning of assignment, the characteristic Ca or Cb chemical shifts of
residues such as Thr/Ser and Gly can help to identify the residue type in the
sequential resonance assignment; proline residues can interrupt the assignment.
However, the residue type identification usually has to be consistent with the
information from 13C-13C or 1H-1H TOCSY-type experiments, or from the pulse
schemes designed for determining the spin topology of specific side-chains and
chemical shifts [23].Nearly complete backbone resonance assignments, including 1HN, 15N, 13CO, 13Ca, 13Cb, and 1Ha, were
achieved for wild-type rat L-PGDS (Fig. 5). The side-chain resonances
were approximately 75% complete. The results of assignments indicated that the
wild-type rat L-PGDS obtained using this expression system was suitable for
determination of 3-D NMR solution structure.
Discussion
In this study, we developed an E. coli system expressing a
rat L-PGDS and its mutants with GB1 as the fusion protein. This system yielded
highly soluble and stable fusion proteins, as the fusion partner is very small
and highly soluble to help target proteins fold correctly. The wild-type rat
L-PGDS and four mutants all obtained high yield expression and folded correctly
in this study. The single bands of these enzymes on non-denatured gel
electrophoresis indicated that they were pure and monomeric proteins. The
results of ESI-MS also indicated that there was no formation of intermolecular
disulfide bonds. On non-denatured gel electrophoresis, wild-type enzyme and C65A mutant migrated in the gel faster than other enzyme mutants [Fig.
1(B)], suggesting that Cys89 and Cys186 residues constructed an intramolecular disulfide linkage. The rat
L-PGDS mutants without intramolecular disulfide linkage might become less
compacted during non-denatured gel electrophoresis, and migrate slowly.
However, if Cys65 and Cys186
residues form a disulfide bond, the wild-type rat L-PGDS would display two
states on non-denatured gel electrophoresis. These results further indicated
that the correct disulfide pairing could be formed using this expression
system, which is necessary for structural study. The Cys186 residue in a flexible C-terminal loop of the enzyme could only form
the disulfide linkage with the Cys89 residue, not with Cys65, and the Cys65 and Cys89 residues did not form the disulfide bond. These results suggested that
Cys89 and Cys186 might be exposed on the surface of rat
L-PGDS molecules to a greater extent than Cys65. This
is in agreement with the structure of mouse L-PGDS [10] and the homology
modeling structure, in which the Cys65 residue exists in the
hydrophobic pocket of the enzyme and an intramolecular disulfide bridge between
Cys89 and Cys186 occurs outside the barrel structure.
However, the enzyme activity of L-PGDS decreased concomitantly with the loss of
the free thiol group of Cys65 and the replacement of Cys65 with Ser/Ala by site-directed mutagenesis led to complete loss of
catalytic activity. The absence of the disulfide bond in rat L-PGDS might allow
a broader range of ligands to fit into the barrel pocket and slightly increases
the affinity of rat L-PGDS for ligands (data not shown).To examine the proper folding of the bacterially produced enzymes
and the effects of removing the disulfide bond on their structures, the
proteins were analyzed by CD spectroscopy. The similarities among CD spectra
recorded on these enzymes indicated that they adopted similar secondary
structures and there were non-significant effects on their conformations after
removing the disulfide bond. The CD spectra recorded on rat L-PGDS proteins
described in this work, compared with those published in previous reports for
native or recombinant proteins, indicated the correct folding of all rat L-PGDS
enzymes prepared according to our strategy, even though these enzymes lack
their natural glycosylation due to their expression in a prokaryotic host. The
secondary structure contents of rat L-PGDS enzymes were similar to those of b-lactoglobulin,
a member of the lipocalin family, estimated from both CD spectra and X-ray
crystallography [24]. There were no significant effects on their secondary
structures after removing the disulfide bond due to correct disulfide pairing,
proper folding, and no forming of intermolecular disulfide linkage.Two-dimensional 1H-15N HSQC
spectrum is a reliable method to judge the suitability of a particular protein
for structural determination with NMR spectroscopy. A well-folded protein would
display a wide dispersion of amide proton chemical shifts in 2-D 1H-15N HSQC spectrum [25]. In this study, the
proper folding of rat L-PGDS and its mutants was examined by 1H-15N HSQC spectrum. The high dispersion of 180
cross peaks in the spectrum was indicative of correct folding and well defined
tertiary structures of rat L-PGDS or its mutants. The effects of disulfide on
protein conformation could be elucidated by overlapping the 1H-15N HSQC spectra of rat L-PGDS and its mutants.
By comparing these spectra, we found that the enzymes with disulfide bonds
adopted almost the same conformations, and the enzymes without disulfide bonds
also adopted similar conformations. However, conformations without disulfide
bonds became less compact. The results of HSQC experiments further confirmed
that the correct disulfide bond was formed and there were no significant
effects on their structures after removing the disulfide bond. Thus, we
selected the wild-type rat L-PGDS, which is closest to the native state, for
the determination of the 3-D solution structure by NMR spectroscopy.Using this expression system, we yielded highly soluble, pure, and
stable proteins of rat L-PGDS and its mutants. However, there are three Cys
residues in wild-type rat L-PGDS. We were compelled to identify if these Cys
residues could form the correct disulfide bond before carrying out the
following studies. The results of non-denatured gel electrophoresis indicated
that Cys89 and Cys186 residues could construct disulfide bonds
correctly, but Cys65 did not contribute to the formation of
disulfide bonds. The ESI-MS results further identified that there were no
intermolecular disulfide bonds in rat L-PGDS and its mutants. The results of CD
and 2-D 1H-15N HSQC experiments indicated that there were
no significant effects on their structures after removal of the disulfide bond.
In conclusion, the expression and purification protocol designed for wild-type
rat L-PGDS is suitable and economic to yield enough properly folded and
monomeric protein with correct disulfide pairing to allow determination of 3-D
solution structure by NMR spectroscopy.
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