Refolding of the Escherichia coli expressed extracellular domainof a7 nicotinic acetylcholine receptor Cys116 mutation diminishes aggregation and stabilizes the b structure Victor I.. Th
Trang 1Refolding of the Escherichia coli expressed extracellular domain
of a7 nicotinic acetylcholine receptor
Cys116 mutation diminishes aggregation and stabilizes the b structure
Victor I Tsetlin1, Natalia I Dergousova1, Ekaterina A Azeeva1, Elena V Kryukova1, Irina A Kudelina1, Elena D Shibanova1, Igor E Kasheverov1and Christoph Methfessel2
1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;
2
Central Research Biophysics, Bayer, Leverkusen, Germany
Heterologous expression of the extracellular domains
(ECDs) of the nicotinic acetylcholine receptor (AChR)
subunits may give large amounts of proteins for studying the
functional and spatial characteristics of their ligand-binding
sites The ECD of the a7 subunit of the homo-oligomeric a7
neuronal AChR appears to be a more suitable object than
the ECDs of other heteromeric neuronal or muscle-type
AChRs The rat a7 ECDs (amino-acid residues 1–210)
were recently expressed in Escherichia coli as fusion proteins
with maltose-binding protein [Fischer, M., Corringer, P.,
Schott, K., Bacher, A & Changeux, J (2001) Proc Natl
Acad Sci USA 98, 3567–3570] and glutathione
S-trans-ferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E.,
Chiodini, F., Bertrand, D., Methfessel, C & Tsetlin, V
(2001) J Biol Chem 276, 15810–15815] However, these
proteins exist in solution mostly as high-molecular mass
aggregates rather than monomers or oligomers In the
pre-sent work it is found that refolding of GST–a7-(1–208)
protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates The C116S mutation in the a7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions This mutation slightly increased the affinity of the protein for a-bungarotoxin (from Kd 300 to
150 nM) Gel-permeation HPLC was used to isolate the monomeric form of the GST–a7-(1–208) protein and its mutant almost devoid of SDS CD spectra revealed that the C116S mutation considerably increased the content of
b structure and made it more stable under different condi-tions The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the a7 ECD in an oligomeric form
Keywords: a7 nicotinic acetylcholine receptor; extracellular domains; expression in Escherichia coli; Cys116 mutation;
CD spectroscopy
Nicotinic acetylcholine receptors (AChRs), belonging to the
family of ligand-gated ion channels, are divided into two
major groups: muscle-type and neuronal receptors The
muscle-type AChR from the electric organ of the Torpedo
ray has been studied most comprehensively It is composed
of five subunits, namely two a subunits, and one b, c, and d
subunits Mammalian muscle AChRs are similar, the only
difference being the presence of e subunits in the mature
receptors instead of c in the fetal versions Neuronal AChRs
are composed of a subunits (a2–a10) and b subunits (b2–
b4), either as hetero-oligomers of a/b combinations or as
homo-oligomers, like pentaoligomeric a7 AChRs (reviewed
in [1–3])
The current ideas on the spatial organization of the whole
family of nicotinic AChRs are based mainly on the electron
microscopy data for the Torpedo AChR The subunits are arranged pseudosymmetrically along the central axis form-ing a channel A general shape of the molecule and its disposition in the membrane were established by electron microscopy two decades ago [4] Recent cryo-electron microscopy data provided a better view of the channel structure and of the extracellular portions In particular, a resolution of 4.6 A˚ has been achieved for the extracellular domain (ECD) moiety of the membrane-bound Torpedo AChR [5] This domain accommodates the binding sites for agonists and competitive antagonists Biochemistry and molecular biology data also suggest that all other nicotinic AChRs should have a structural/functional organization similar to that of the Torpedo AChR (reviewed in [1–3]) In principle, heterologous expression of ECDs may provide sufficient amounts of proteins necessary for establishing their high-resolution spatial structure with the aid of X-ray analysis or NMR
The ECD (amino-acid residues 1–210) of the mouse muscle and Torpedo a subunits were obtained by heterolo-gous expression in mammalian cells [6] and in E coli [7–9], respectively The secondary structure of these proteins was determined by CD spectroscopy However, these proteins
a p riorilack the contacts with other non-a subunits that also participate in forming the ligand-binding sites in the intact receptors This drawback has been overcome by expressing the ECDs of all Torpedo subunits that assemble into a
Correspondence to V I Tsetlin, Shemyakin-Ovchinnikov
Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 Miklukho-Maklaya Str., V-437 Moscow GSP-7, 117997 Russia.
Fax/Tel.: + 7095 335 57 33, E-mail: vits@ibch.ru
Abbreviations: AChR, acetylcholine receptor; AChBP,
acetylcholine-binding protein; ECD, extracellular domain; MBP, maltose-acetylcholine-binding
protein; GST, glutathione S-transferase; aBgt, a-bungarotoxin; IPTG,
isopropyl thio-b- D -galactoside; GdnHCl, guanidine hydrochloride.
(Received 6 December 2001, revised 8 April 2002,
accepted 29 April 2002)
Trang 2pentaoligomer in a baculovirus system [10].
of the domain approach became even more clear after
solving the X-ray structure of the acetylcholine-binding
protein (AChBP) from Lymneae stagnalis
breakthrough in the field This water-soluble protein has the
size of the AChR subunit ECD and forms homopentamers
In view of this finding, it seems especially interesting to
prepare for structural analysis the ECD of the a7 AChR, i.e
of the receptor known to function as a homopentamer [13]
In fact, there are already several publications on the a7 ECD
heterologous expression The chicken a7 ECD (including
also the transmembrane fragment M1) was isolated after
expression in Xenopus oocytes [14] It formed an oligomer,
but the protein was obtained only in minute amounts Large
amounts of the rat a7 ECD (residues 1–196) were obtained
recently in E coli as a water-soluble fusion protein with the
N-terminally attached maltose-binding protein (MBP) [15]
However, the major obstacle for structural studies appears
to be the presence of aggregates with molecular masses
considerably exceeding that of pentaoligomers A similar
problem was encountered in our laboratory with rat a7
ECD (residues 1–208) fused to glutathione S-transferase
(GST) [16] or to MBP
Here we report optimization of refolding conditions,
which for the GST–a7-(1–208) protein resulted in the
diminished tendency for aggregation The aggregation
could be further decreased and, under certain conditions
completely prevented, by blocking free SH group(s) in the
refolded protein or by mutating the Cys116 residue It
allowed us to obtain the monomeric forms at relatively high
concentrations This more stable C116S monomer appears
to be useful for structural studies and may serve as an
intermediate on the way to an oligopentamer As a first step,
we determined its secondary structure by CD spectroscopy
and found a predominance of b structure, similar to what
was found in the extracellular moiety of the intact Torpedo
AChR [5], muscle a subunits [6–9] and what is characteristic
of AChBP [12]
E X P E R I M E N T A L P R O C E D U R E S
Construction of expression vectors
The gene encoding the rat a7 AChR ECD was amplified by
PCR using the a7 cDNA cloned in the pBS SK(–) (provided
by H.-J Kreienkamp) as template and primers 1/2 for
GSTa7-(1–208) and 5/6 for MBP-a7-(1–208) (see Fig 1)
The PCR products were digested with appropriate
restric-tion enzymes (BamHI and HindIII for GST fusion proteins;
XmnI and BamHI for MBP fusion protein), gel-purified
using the QIAquick gel extraction kit (Qiagen) and ligated
with linearized pGEX-KG vector (Amersham Pharmacia
Biotech) for GST fusion proteins and pMAL-c2 vector
(New England Biolabs) for MBP-a7-(1–208) The ligation
reaction mixtures were used to transform E coli cells
JM109 for GST fusion proteins and TB1 cells for
MBP-a7-(1–208) Potential clones were screened with colony PCR,
and the presence of the insert was confirmed by restriction
analysis
To obtain the C116S mutant of the fusion protein GST–
a7-(1–208), designated GST–a7m-(1–208), site-directed
mutagenesis was performed in a two step PCR The
forward mutagenic primer 3 (see Fig 1) contained an SspI
restriction site The codon CAG for Gln117 was exchanged for CAA and the codon CTC for Leu119 for TTG to generate an SspI restriction site The reverse primer 4 also contained an SspI restriction site Each construct was verified by DNA sequencing
Proteins expression, purification and refolding GST fusion proteins JM109 cells carrying appropriate constructs were grown in Luria–Bertani medium with ampicillin (100 lgÆmL)1) at 37C (about 3 h) When the
D600 reached a value of 0.4–0.6, isopropyl thio-b-D-galactoside (IPTG) was added to final concentration
of 0.3 mM, and the bacteria were further cultured for 3 h at
37C Cells were pelleted by centrifugation (15 min,
10 000 g) and stored at )20 C Both fusion proteins GST–a7-(1–208) and its C116S mutant GST–a7m-(1–208) were found in the inclusion bodies The cells were suspended
in 10 mMTris/HCl, 150 mMNaCl, 1 mMEDTA, pH 7.5, and then disrupted by sonication (10 pulses by 30 s, 10C) After centrifugation (10 min, 14 000 g), the inclusion bodies were washed intensively three times with the above buffer containing in a series 0.1% Triton X-100, 2MNaCl, and then 2M urea At each washing step the pellet was resuspended by sonication Finally, the pellet was washed with 10 mMTris/HCl, pH 7.5, harvested by centrifugation (15 min, 20 000 g) and stored at)20 C Partially purified inclusion bodies were solubilized in 50 mM Tris/HCl, 8M
urea or 6M GdnHCl, 10 mM dithiothreitol at room temperature, with gentle stirring overnight The concentra-tion of fusion protein at this step was no more than
1 mgÆmL)1 Denaturing and reducing agents were removed
by dialysis against 20 mMTris/HCl, pH 8.0, at 10C, 24 h Three different conditions were tested for refolding of the GST fusion proteins: in the presence of 0.1% Chaps, 0.1% SDS, or without any detergent in the protein solution and in dialysis buffer The concentration of each detergent was
Fig 1 Schematic representation of recombinant fusion proteins con-taining the rat a7 ECD The positions of the disulfide bridges in GST and a7 ECD, as well as of the unpaired Cys116 in a7, are indicated under the rectangles 1–6, primers used for preparing the respective cDNAs (reconstituted restriction enzyme recognition sites are in bold, italics indicate the mutation sites).
Trang 3below critical micellar concentration Oxidation of the fully
reduced protein was performed on air
monitored by titration of free SH groups [17] If a precipitate
was formed, it was removed by centrifugation (15 min,
20 000 g), and the protein solution was stored at 4C
GST was expressed in E coli strain BL21 as a soluble
protein, and was purified by chromatography on
glutathi-one–agarose as recommended by manufacturer (Amersham
Pharmacia Biotech) The purified protein was denatured in
50 mM Tris/HCl, 8M urea, 10 mM dithiothreitol and
refolded under the conditions described above
MBP fusion protein Different E coli strains were used to
optimize the MBP-a7-(1–208) expression The best results
were obtained with a protease-deficient strain CAG597 The
cells with recombinant plasmid were grown in Luria–
Bertani medium with 0.2% glucose and ampicillin
(100 lgÆmL)1) at 37C (about 6 h) IPTG was added to
final concentration of 0.3 mM (D600¼ 0.4–0.6), and the
bacteria were further cultured overnight at 30C Cells were
pelleted by centrifugation (10 min, 10 000 g) and stored at
)20 C MBP-a7-(1–208) was expressed mostly as a soluble
protein, smaller amounts being detected by SDS/PAGE in
the inclusion bodies
MBP-a7-(1–208) was purified by chromatography on
amylose resin as recommended by manufacturer (New
England Biolabs), using 20 mM maltose for eluting the
fusion protein After purification, the protein obtained was
unstable However, it could be stored for over a month at
4C in the following buffer: 20 mM Tris/HCl, pH 7.5,
200 mM NaCl, 1 mM EDTA, 0.05% octyl-b-D-glucoside,
1 mM sodium azide, 1 lM pepstatin A, 10 lM leupeptin,
10 lMchymostatin, 10 lMantipain, 10 lMbestatin, 1 mM
phenylmethanesulfonyl fluoride
MBP was expressed and purified under the same
conditions
With the same protocol, the expression of the C116S
mutant of MBP-a7-(1–208) was carried out to give mostly a
soluble protein However, in contrast to all other expressed
products, this mutated protein was found considerably
more toxic for E coli and the level of expression was very
low; insufficient amounts of the purified MBP-a7m-(1–208)
protein were obtained to perform further studies
Determination of protein concentration
Protein concentrations were determined using the Bradford
Protein assay (Bio-Rad) with bovine serum albumin as
reference, and by UV spectra Extinction coefficients
(k 278 nm) for each fusion protein were determined as a
sum of all the extinction coefficients of the protein aromatic
amino acids
SDS/PAGE and Western blotting
All protein samples were analyzed by SDS/PAGE according
to Laemmli [18] in a 12% gel Samples were prepared under
denaturating reducing conditions (by boiling in a sample
buffer containing 1% 2-mercaptoethanol and 2% SDS) or
under nonreducing conditions (without boiling and with no
2-mercaptoethanol in a sample buffer containing 0.1%
SDS) For Western blotting, the proteins were transferred
from unstained SDS gels to a nitrocellulose membrane using
a TransBlot SemiDry Electrophoretic Transfer (Amersham Pharmacia Biotech) at 30 V in 2 h After blocking with 1% bovine serum albumin in phosphate buffered saline, pH 8.2, membranes were incubated with125I-labelled a-cobratoxin from the Naja kaouthia cobra venom at 10C overnight Unbound toxin was removed by washing, and labeled proteins were detected by autoradiography
Gel-permeation HPLC Gel-permeation HPLC was carried out on a Super-dex 200 HR column (10· 300 mM; Amersham Pharmacia Biotech) by isocratic elution at a flow rate 0.5 mLÆmin)1in
20 mMTris/HCl buffer, pH 8.0, containing 150 mMNaCl, either in the presence of 0.1% detergent (SDS or Chaps) or
in the absence of any detergent The column was calibrated with standard proteins dissolved in the same buffers as the expressed proteins under examination
Binding experiments
125I-Labelling of a-bungarotoxin (aBgt) and a-cobratoxin (for Western blotting experiments) was carried out with the chloramine T method followed by desalting on a G15 Sephadex column in 50 mM Tris/HCl buffer, pH 8.0, as described previously [19] Equilibrium binding was analyzed
on anion-exchange filters DE81 [20] in a fast filtration modification [21] Various amounts of 125I-labelled aBgt (from 1.5 to 70 pmol, specific radioactivity 25 CiÆmmol)1) were incubated with 20 pmol of different proteins for 2 h at room temperature in 50 lL of 50 mM Tris/HCl buffer,
pH 8.0 The final concentration of detergents in the reaction mixtures was 0.004 or 0.06% for the proteins refolded in 0.1% SDS or 0.1% Chaps, respectively The SDS concen-tration was determined in reaction with p-rosaniline chloride [22] Nonspecific binding was determined by preincubation for 1 h of the expressed products with a 100-fold molar excess of a-cobratoxin isolated from the Naja kaouthia cobra venom As the additional controls, binding experi-ments with the expressed GST or MBP were carried out under the same conditions Incubation mixtures were applied on DE81 filters presoaked in 50 mM Tris/HCl buffer, pH 8.0, containing 0.1% Triton X-100, and quickly washed under vacuum with 5 mL of the same buffer The analysis of binding experiments was carried out using
ORIGINv5.0 (MicroCal Software, Inc.)
GST–a7-(1–208) fusion protein modification withN -ethylmaleimide
A 0.5-mL volume of refolded fusion protein GST–a7-(1–208) solution (0.2 mgÆmL)1) in 20 mMTris/HCl, pH 8.0, was incubated with 10 mM dithiothreitol for 3 h at room temperature The reduced protein was dialyzed against the same buffer without dithiothreitol for 20 h at 4C and oxidized by air A decrease in the free SH content was monitored by the Ellman’s method The protein solution containing 1.0 ± 0.1 SH per mol protein was obtained N-ethylmaleimide was added to a final concentration of
1 mM, incubated for 15 min at room temperature, and then the excess of N-ethylmaleimide was removed by dialysis The residual content of SH groups was found to be less than 0.15 mol SH per mol protein
Trang 4CD spectroscopy
CD spectra were recorded on a JASCO J-500A
spectro-polarimeter (Japan) The results were expressed as molar
ellipticity, [Q] (degÆcm2Ædmol)1), with the average mean
amino-acid residue weight (MRW) of 115 The molar
ellipticity was determined as [Q] ¼ Q · 100MRWÆc)1Æl)1,
where c is the protein concentration in mgÆmL)1, l is the
light path length in centimeters, and Q is the measured
ellipticity in degrees at a wavelength k The instrument was
calibrated with (+)-10-camphorsulfonic acid, assuming
[Q]291¼ 7820 degÆcm2Ædmol)1 [23] Secondary structure
was calculated according to the program CONTIN for
globular proteins [24]
R E S U L T S
Expression inE coli and refolding
of the GST– a7-(1–208) protein, its C116S mutant,
and MBP-a7-(1–208) protein
These fusion proteins are schematically depicted in Fig 1
Figure 2 shows the SDS/PAGE analyses in reducing
conditions of the (GST)-a7-(1–208) (lane 1) and of the
respective C116S mutant (lane 2) after refolding from
inclusion bodies in the presence of 0.1% SDS A similar
picture was observed when these proteins were refolded
either in the presence of 0.1% Chaps or in aqueous buffer
without any detergents (data not shown) The 1–208
fragment was also successfully expressed as a soluble
MBP-a7-(1–208) protein and isolated with the aid of affinity chromatography on an amylose resin (lane 3) A compar-ison with the standards (lane 4) shows that all the proteins obtained have apparent molecular masses in the expected range
On storage of the GST–a7-(1–208) protein refolded in the absence of any detergents, precipitation usually occurred This protein could be kept in solution in concentrations up
to 1.2 mgÆmL)1if supplemented with 0.1% Chaps [16] Somewhat higher concentrations ( 1.5–2.0 mgÆmL)1) were achieved with refolding in the presence of 0.1% SDS The solutions in 0.1% SDS-containing buffers could
be stored at 4C for over a month, the C116S mutant being especially stable Although the MBP-a7-(1–208) was pro-duced as a water-soluble protein, after purification it could
be kept for prolonged period in solution at concentrations below 0.2 mgÆmL)1only if supplemented with 0.05% octyl-b-D-glucoside and a cocktail of protease inhibitors Analysis of a-bungarotoxin binding
As seen from Fig 3A, the expressed proteins specifically bind 125I-labelled a-cobratoxin on blots Data on the
125I-labelled aBgt equilibrium binding in solution are compiled in Table 1 and illustrated in Fig 3B for GST– a7-(1–208) and its C116S mutant The Kdvalues are in the range of 100–820 nMand depend on the refolding condi-tions When refolded in the presence of 0.1% SDS, the GST–a7-(1–208) and its C116S mutant have very similar Kd
values (310 and 160 nM, respectively) The difference was larger (180 and 820 nM) when refolding was carried out in 0.1% Chaps For MBP-a7-(1–208) protein, Kdvalues were
in the low micromolar range (data not shown)
It is known that aBgt binds to the full-size a7 AChR in the low nanomolar range [13,25] A Kdof 1.6–2.0 nMwas reported for the extended chicken a7 ECD expressed in the Xenopus oocytes [14], whereas a water-soluble protein MBP-a7-(1–196) produced in E coli had a Kdof 2.5 lM
[15] The highest affinity for GST–a7-(1–208) proteins was
150 nM, which is still considerably weaker than for the full-size a7 AChR
4 However, the GST–a7-(1–208) refolded
in 0.1% Chaps was able to distinguish the long-chain a-neurotoxins from the short ones, as well as the a7 AChR targeting a-conotoxin ImI from the muscle-type targeting a-conotoxin GI [16] These properties of the intact a7 AChRs are preserved in the GST–a7-(1–208) refolded in 0.1% SDS (data not shown)
Analysis of aggregation state The aggregation state of the fusion proteins containing the rat a7 ECD was examined by SDS/PAGE (Fig 4) and gel-permeation HPLC (Fig 5) In the presence of 2-mercapto-ethanol, the GST–a7-(1–208) protein refolded in 0.1% SDS
is predominantly a monomer, whereas in the absence of the reducing agent it contains also a considerable portion of oligomers and high-molecular mass aggregates (cf lanes 1 and 2 in Fig 4)
For the chick a7 AChR heterologously expressed in a mammalian cell line, it was recently shown that cysteines of the ECD are involved in aggregation, which can be considerably decreased by reducing agents or by treatment with N-ethylmaleimide [26] We decided to check whether
Fig 2 SDS/PAGE of recombinant fusion proteins containing the rat a7
ECD (1 and 2) GST–a7-(1–208) and GST–a7m-(1–208) fusion
pro-teins, respectively, after refolding from the inclusion bodies in the
presence of 0.1% SDS; (3) MBP-a7-(1–208) fusion protein after
affinity purification on amylose resin; (4) molecular mass markers (in
kDa).
Trang 5modification with N-ethylmaleimide gave a similar effect for
the rat a7 ECD expressed in E coli and the consequences of
deleting the SH group of the specified residue, Cys116, by
mutating the latter into a Ser
After reduction–reoxidation of the GST–a7-(1–208) and
dialysis, only 1 SH group per mol was detected by Ellman’s
reagent Because of the absence of free SH groups in GST
(see Fig 1), there were reasons to believe that it was that of
Cys116 The N-ethylmaleimide treatment of the fusion
protein GST–a7-(1–208) refolded in 0.1% SDS leads to an
clear decrease in aggregates and oligomers on nonreducing
gels (cf lanes 2 and 6 in Fig 4) The effect of mutating
Cys116 is even more dramatic: there is almost no difference
between the SDS/PAGE under reducing and nonreducing
conditions (lanes 3 and 4 in Fig 4), the monomer prevailing
in both cases This result explains why the solutions of the
C116S mutant in 0.1% SDS are much less prone to aggregation and precipitation on prolonged storage as compared to the GST–a7-(1–208) protein itself
The dependence of the aggregation state on the refolding conditions and on the introduced mutation was examined
by gel-permeation HPLC on a Superdex 200 column (Fig 5) The GST–a7-(1–208) proteins, refolded in the presence of 0.1% SDS, were subjected to chromatography
in the presence of 0.1% SDS, which is similar to SDS/ PAGE under nonreducing conditions It was found that the fraction of aggregates (marked with an asterisk) is consid-erably smaller for the C116S mutant (Fig 5A) The major broad peak centred at 70 kDa originates mainly from a monomeric fraction as indicated from its position on SDS/ PAGE under nonreducing conditions (data not shown; the shoulders at < 43 kDa are the concomitant E coli pro-teins) The consequences of mutation are very pronounced for the GST–a7-(1–208) proteins refolded in 0.1% Chaps Chromatography in a buffer containing the same detergent showed in the GST–a7-(1–208) protein a large peak of aggregates overlapping the oligomeric and monomeric fractions, while for the C116S mutant a broad peak of oligomers dominated (Fig 5B)
The proteins obtained were also analyzed on a Super-dex 200 column equilibrated in purely aqueous buffers For the GST–a7-(1–208) protein refolded in Tris/HCl buffer containing 1 mM dithiothreitol and dialysed against the same buffer without dithiothreitol, the intense peak of high-molecular mass aggregates (m > 600 kDa) and a broad peak with a centre at 450 kDa corresponding to oligo-mers and monooligo-mers are present (Fig 5C) It should be noted that the resolution of the column is better here than in the presence of 0.1% SDS, the standards on average elute later (cf Fig 5A,C) For the GST–a7m-(1–208) protein, also refolded in the absence of 0.1% SDS, the peak of aggregates is decreased by 25%, the centre of broad peak
of oligomers shifts from 450 to 300 kDa (corresponding to oligomers of five or six units) When the MBP-a7-(1–208) protein, produced in a water-soluble form and not subjected
to action of such denaturants as 8Murea or GdnHCl, was chromatographed under the conditions of Fig 5C, only one sharp peak of high-molecular mass aggregates was obtained (Fig 5D) showing that this protein is not promising for further physicochemical studies On the other hand, the chromatography on Superdex 200 in the absence of 0.1% SDS revealed a dramatic difference between the GST–a7-(1–208) protein and its C116S mutant when they were refolded in the presence of 0.1% SDS (Fig 5E) Whereas for GST–a7-(1–208) the peak of aggregates is much larger than the broad peak centred at 70 kDa, the latter is predominant for GST–a7m
Thus, the presented SDS/PAGE and gel-permeation HPLC data show that the problem of aggregation can be partly solved by chemical modification of accessible sul-fhydryl groups or by mutating the Cys116 residue How-ever, formation of intermolecular disulfides with the participation of the Cys116 sulfhydryl is one of the factors leading to aggregation, but not the sole one A further decrease in the aggregation extent depends only weakly on the state of SH groups and requires the addition of 0.1% SDS
The monomeric fractions 2 (Fig 5E) were collected for further analysis The K values characterizing their
inter-Fig 3 Interaction of fusion proteins containing a7 ECD with iodinated
a-neurotoxins (A) Autoradiography of 125I-labelled a-cobratoxin
binding by MBP-a7-(1–208) (lane1) and GST–a7-(1–208) (lane 2) on
blots Lane 3, protein standards as detected by Coomassie staining of
the respective portion of the gel (B) 125 I-Labelled aBgt binding curves
for GST–a7-(1–208) protein (filled circles) and GST–a7m-(1–208)
protein (open circles) refolded in 0.1% SDS Before the binding assay,
the SDS concentration was decreased to 0.004% by dilution with
aqueous buffer The indicated K d values are averaged from two
inde-pendent experiments and calculated using ORIGIN 5.0.
Trang 6action with125I-labelled aBgt (see Table 1) are very close to
those of the starting GST–a7-(1–208) and
GST–a7m-(1–208) proteins, confirming a somewhat higher affinity of
the C116S mutant The SDS content in the pooled fractions
in the reaction with p-rosaniline chloride [22] was estimated
to be 0.0001% When these fractions were reapplied to the
same column equilibrated without 0.1% SDS, only the
peaks of monomers were observed, virtually without traces
of aggregates (Fig 5F, fraction 2) For comparison,
rechromatography of the pooled aggregates peaks is shown
(Fig 5F, fraction 1)
CD spectra analysis
CD spectra of the GST–a7-(1–208) protein and its C116S
mutant look similar (Fig 6), having the contributions from
both a helices and b structure
(Table 2) revealed that the content of the secondary structure varies depending on the protein and the refolding conditions In general, these variations are smaller for the C116S mutant: the secondary structure is very similar for the protein refolded either in purely aqueous solution, or refolded and analyzed in 0.1% SDS, or isolated from the latter by HPLC in the absence of detergent The average content of the a helices, b structure and unordered confor-mation is estimated to be 22, 45, and 33%, respectively It is also clear from the Table 2 that under all similar conditions, the C116S mutant is characterized by almost a twofold higher content of b structure than the starting GST–a7-(1–208) protein In contrast, the latter has a 1.5-fold higher content of a helices and a somewhat higher percentage of unordered structure In view of the more stable secondary structure of the mutant and its decreased tendency to aggregation, we assume that the C116S mutant is a better model of the monomeric a7 ECD than the starting GST– a7-(1–208) protein If we further assume that GST, which does not change its secondary structure under different conditions (Table 2), also preserves it in the a7 ECD fusion proteins, a rough estimate of the secondary structure can be made for the a7 ECD moiety Because GST and a7 ECD have very close molecular masses, their contributions to CD curves should be almost equal Using the averaged values for the C116S mutant, we obtained the following values for the a7 ECD: 17% a helix, 56% b structure and 27% of unordered structure
D I S C U S S I O N
The results obtained, along with other recently reported data [15,16], show that ECD of the rat a7 AChR can be heterologously expressed in E coli as different fusion proteins soluble, either in purely aqueous solutions or in the presence of detergents The respective proteins bind aBgt and other long-chain a-neurotoxins with an affinity which is not strongly dependent on the chosen variant of the a7 ECD, production of proteins in water-soluble form or their recovery from the inclusion bodies, or on refolding with or without detergents The earlier reported affinities for MBP-a7-(1–196) in aqueous solution [15] and for
GST–a7-Table 1 Radioligand assay data of 125 I-labelled iodinated a-bungarotoxin binding by the proteins refolded under different conditions.
Protein
Detergent content in refolding buffer (%)
125
I-Labelled aBgt binding parameters
K d (n M ) B max (%) b
a The monomeric forms were prepared by ultrafiltration of the HPLC purified monomeric fractions (see Fig 5E,F) using an Amicon 8010 ultrafiltration membrane YM10 SDS content in the concentrated samples was determined in reaction with p-rosaniline chloride [22].bThe
B max values are presented as a percentage ratio of the calculated B max in n M to the concentration of the respective protein (in n M ) in the incubation mixture The data presented were calculated with the use of ORIGIN 5.0.
Fig 4 Analysis of the a7 ECDs aggregation state by SDS/PAGE in the
presence (+) or absence (–) of 1% 2-mercaptoethanol (ME) Lanes 1
and 2, GST–a7-(1–208); lanes 3 and 4, GST–a7m-(1–208) mutant;
lanes 5 and 6, GST–a7-(1–208) modified with N-ethylmaleimide, lane
7, molecular mass markers, kDa.
Trang 7(1–208) in 0.1% Chaps [16] are in the low micromolar range.
The Kdvalues obtained in this work are in the range of 100–
850 nM, the highest affinity being shown by the C116S
mutant of GST–a7-(1–208) protein refolded from the
inclusion bodies in the presence of 0.1% SDS (Table 1)
Although even this affinity is much weaker than that of the
intact a7 AchR, which binds aBgt with K 1–5 nM
[13,25], such important features of the a7 AChR selectivity
as the capacity to discriminate long-and short-chain a-neurotoxins and various a-conotoxins, are retained in the expressed a7 ECD (see Results)
High molecular mass aggregates, whose molecular mas-ses are considerably larger that of a pentamer, are the major obstacle to obtainaing soluble and correctly folded hetero-logously expressed ECDs of AChRs Aggregation usually leads to insoluble precipitates However, a7 ECDs may contain high molecular mass aggregates even in solution (Fig 5) The major cause of poor solubility might have been the necessity of isolating the proteins from inclusion bodies under denaturing conditions and then to refold them Therefore, much effort has been made to produce the AChR ECDs or their large portions as soluble fusion proteins [15,27
7 ] In particular, this goal was achieved for the rat a7 ECD in [15] and in the present work using similar fusion proteins with MBP However, these soluble proteins contained various amounts of aggregates
Aggregation and precipitation of the proteins obtained from the inclusion bodies could be partly overcome by optimizing the refolding conditions If 0.1% SDS was present from the stage of dissolving the inclusion bodies, the GST–a7-(1–208) protein could be kept in solution (Tris/ HCl buffer, pH 8.0, 0.1% SDS) for a long time at concentrations about 1 mgÆmL)1 Under these conditions the major fraction of the protein is a monomer, but the fraction of high-molecular mass aggregates is also quite large (Fig 5A) It is known that SDS is capable of inhibiting the aggregation of bacterially expressed or denatured proteins and therefore is widely used in refolding studies [28–30] It is assumed that the masking of hydrophobic protein interfaces by detergent molecules results in
reduction in aggregation There are also indications that SDS diminishes the aggregation by inhibiting the formation
Fig 5 Gel-permeation HPLC of the a7 ECD-containing fusion proteins
on a Superdex 200 HR column (A) GST–a7-(1–208) (solid line) and
GST–a7m-(1–208) (dashed line) refolded in the presence of 0.1% SDS
are analyzed on the column equilibrated in 20 m M Tris/HCl, pH 8.0,
containing 150 m M NaCl (elution buffer) supplemented with 0.1%
SDS Proteins (30–50 lg) were eluted at a flow rate of 0.5 mLÆmin)1.
The same conditions, with the exception of presence or absence of
detergents in the elution buffers, and designations apply to (B), (C) and
(E) (B) GST–a7-(1–208) and GST–a7m-(1–208) refolded in the
pres-ence of 0.1% Chaps, the column is equilibrated and run in the elution
buffer containing 0.1% Chaps (C) GST–a7-(1–208) and
GST–a7m-(1–208) proteins refolded in the absence of detergents, the elution
buffer is without detergents (D) MBP-a7-(1–208) protein analyzed in
the conditions of (C) (E) GST–a7-(1–208) and GST–a7m-(1–208)
proteins refolded in the presence of 0.1% SDS are analyzed as in (C)
and (D) (F) Rechromatography of the fractions marked with bars 1
(aggregates) and 2 (monomer) collected on chromatography of GST–
a7-(1–208) (E) In this figure solid and dashed lines correspond to
chromatography of the fractions 1 and 2 from (E), respectively In all
figures, vertical short lines correspond to the elution times of protein
standards (with masses in kDa) under the chosen chromatographic
conditions, the asterisk marking the exclusion volume.
Fig 6 CD spectra of fusion protein GST–a7-(1–208) (solid line) and GST–a7m-(1–208) mutant (dashed line) refolded in 20 m M Tris/HCl,
pH 8.0, containing 0.1% SDS.
Trang 8of intermolecular disulfide bridges [31] Note that
aggrega-tion, oligomerization and the acquisition of aBgt binding
capacity by the a7 and Torpedo AChRs were shown by
Green and coworkers to depend on the state of cysteine
residues in the ECDs of the a7 and a subunits, respectively
[26,32] The major role of the redox state of the disulfide
Cys128–Cys142 was assumed for the a7 AChR [26],
whereas the involvement of the disulfide Cys192–Cys193
was demonstrated for the Torpedo a subunit [32] The a7
ECD has the Cys128–Cys142 disulfide bond
for the whole family of ligand-gated ion channels, as well as
the vicinal disulfide bond
present also in all other a subunits of neuronal and
muscle-type AChRs On the other hand, Cys116 is present
only in a7 and a8 subunits We thought that the presence of
a free SH group of Cys116 might be one of the factors
leading to formation of intermolecular disulfides and
aggregates in the bacterially expressed a7 ECDs
Indeed, the GST–a7m-(1–208) protein on SDS/PAGE
under nonreducing conditions was present mainly as a
monomer, whereas the starting protein contained a large
proportion of aggregates (Fig 4) A similar effect could be
achieved by blocking free sulfhydryl groups in
GST–a7-(1–208) protein by N-ethylmaleimide (Fig 4) The
consid-erable decrease in aggregation caused by the C116S
mutation with the proteins refolded and analyzed in the
presence or absence of various detergents is illustrated by
HPLC data (Fig 5) For the proteins refolded in the
presence of 0.1% SDS, chromatography on a column
equilibrated in a purely aqueous buffer allowed us to obtain
the monomeric fractions (Fig 5E,F) These fractions bind
125I-labelled aBgt with the affinities similar to those of the
starting proteins (Table 1) Because the mutant revealed
even higher affinity, the presence of the Cys116 free SH
group is not essential for aBgt binding
CD spectra (Fig 6 and Table 2) indicate that the GST–
a7-(1–208) and GST–a7m-(1–208) proteins refolded under
different conditions contain varying amounts of regular
secondary structure Interestingly, under all similar
condi-tions the C116S mutant has about a twofold higher content
of b structure than the GST–a7-(1–208) protein The C116S
mutant is less prone to aggregation (see Figs 4 and 5), binds
aBgt even better than the starting protein (Table 1), and its
CD curves are not very sensitive to the conditions of
refolding or measuring the spectra Therefore, the confor-mation of the a7 moiety in the mutant may be more similar
to the ECD conformation of the intact a7 AChR Although calculation of the secondary structure of the 1–208 fragment from the CD data for the fusion protein GST–a7-(1–208) and GST might seem arbitrary, we believe that it gives a qualitatively correct conclusion: the measured relatively high content of b structure is not largely due to the contribution of GST
A high content of b structure found experimentally for the C116S mutant and the even higher content calculated for the a7 ECD moiety is important It is presumed that the ECDs in different, hetero-oligomeric or homo-oligomeric AChRs, have a similar spatial organization CD analyses of the mouse muscle a subunit ECD heterologously expressed
in mammalian cells [6] and of the Torpedo a subunit ECD expressed in E coli [7–9] gave an estimate of 50% for
b structure These results are in good agreement with the subsequently published data of cryo-electron microscopy, which revealed a high content of b structure in the ECD of the intact Torpedo californica AChR [5] Our present results suggest that b structure is an important element of spatial organization of the a7 ECD
When our work was in progress, isolation of acetylcho-line-binding protein (AChBP) from a mollusc Lymneae stagnalis and X-ray analysis of the respective protein heterologously expressed in yeast have been published [11,12] This protein, with 24% homology to a7 ECD, contains practically all amino-acid residues of the AChR ligand-binding sites, has two invariant disulfides and lacks extra Cys116 of a7 It has an immunoglobulin-like topology (rich in b structure) and forms homopentamers [12] There-fore, at least in terms of secondary structure, the monomeric form of the GST–a7m-(1–208) protein resembles the protomer of the AChBP This would justify further efforts
to prepare the oligomeric a7 ECD using the obtained monomers as the starting material
A C K N O W L E D G E M E N T S
The authors are grateful to Dr H.-J Kreienkamp for a7 cDNA clone, to
O Ustinova for help with CD measurements and to Dr J Freigang for fruitful discussion The work was supported by grants (to V T.) from Bayer AG (Leverkusen) and Russian Foundation for Basic Research.
Table 2 CD data of the different expressed proteins refolded under various conditions.
Protein
Detergent content in refolding buffer
Concentration (mgÆmL)1)
Calculated secondary structure (%)
a See the respective note in the Table 1.
Trang 9R E F E R E N C E S
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