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Keywords:

  • expression in E. coli;
  • MALDI mass spectrometry;
  • nicotinic acetylcholine receptor;
  • Torpedoα-subunit domain

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of α2βγδ subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1–209 of the Torpedo californica AChR α-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1–209 sequence, were used and the recombinant proteins obtained (designated α1-209pET and α1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]α-bungarotoxin with equal potency (KD≈ 130 nm, Bmax≈ 10 nmol·mg–1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly β-structure (50–56%), the fraction of α-helices amounting to 32–35%. Nonionic (β-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of β-structure or increased the α-helicity, respectively. The predominance of β-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR α-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem.272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR α-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Abbreviations
αBgt

α-bungarotoxin

AChR

nicotinic acetylcholine receptor from Torpedo californica

FTIR

Fourier-transformed infrared spectroscopy

IPTG

isopropyl β-d-thiogalactopyranoside

MALDI

Matrix assisted laser desorption ionization

NTA

nitrilo-tri-acetic acid

TFE

trifluoroethanol

The nicotinic acetylcholine receptor (AChR) is a member of the superfamily of ligand-gated ion channels. The abundant AChR from the Torpedo electric ray was the first neurotransmitter receptor to be extensively characterized by biochemical, biophysical and electrophysiological methods (see reviews in [1–3]). The molecule is composed of five subunits with the stoichiometry α2βγδ. According to the current model of the AChR organization, each subunit consists of extracellular and intracellular domains with four transmembrane regions (M1–M4). The major part of the extracellular domain is the N-terminal fragment of about 200 amino acid residues, but there is also a loop between the M2 and M3 regions and a short C-terminal tail. The intracellular domain consists of a short loop between the M1 and M2, and a long loop joining M3 and M4, which is important for clustering of AChR molecules and for their attachment to the cytoskeleton [4]. The ion channel is formed principally by the M2 regions of all five subunits [5,6], but data indicate also possible involvement of the M1 transmembrane sequences [7,8]. The two α-subunits play the major part in the formation of ligand-binding sites. However, a large body of evidence indicates that the adjacent γ- and δ-subunits contribute to the binding sites in the intact molecule: low molecular weight agonists and antagonists, like d-tubocurarine, as well as the α-conotoxins and snake venom α-neurotoxins, bind at the α/γ- and α/δ-interfaces [9–13]. The overall shape of Torpedo AChR was determined by electron microscopy at the end of the 1970s [14], before the primary structure of the receptor subunits was known. Subsequently, using tubular receptor preparations and cryo-electron microscopy, Unwin increased the resolution to the extent that structural elements such as possible transmembrane rods in each subunit [15] and α-helices in the extracellular domains could be visualized [16]. However, the lack of AChR crystals suitable for X-ray analysis has precluded further refinement of the AChR spatial structure.

The structural analysis of Torpedo AChR could be reduced, as a first approximation, to the study of its domains. The N-terminal fragment (amino acid residues 1–209) of the α-subunit is important because of its role in ligand binding and because the isolated α-subunit, or synthetic peptides corresponding to the α-subunit region 180–200, bind α-bungarotoxin (αBgt), although with considerably lower affinity than that for the intact AChR [17–19]. Binding of αBgt to the recombinant N-terminal domain of the Torpedoα-subunit or its fragments has also been demonstrated [20–22], but it is not clear how well the recombinant polypeptides reproduced the conformation of the corresponding sequences in the intact molecule. In this paper we describe the expression in Escherichia coli of two polypeptides covering the N-terminal extracellular domain (amino acid residues 1–209) of the Torpedo californicaα-subunit, their purification by metal chelate chromatography and characterization using reverse-phase HPLC, Edman sequencing and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The proteins bound αBgt with high affinity and also bound monoclonal antibodies previously raised against intact Torpedo AChR. Binding of αBgt could be suppressed by d-tubocurarine and decamethonium bromide. Circular dichroism (CD) spectra demonstrated the predominance of β-structure. This result, which agrees with the recent CD data for a similar AChR mouse muscle domain expressed in mammalian cells [23], indicates that β-structure contributes substantially to the conformation of the extracellular domain of the intact AChR.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Materials

All enzymes were from Boehringer Mannheim, Germany. The following vectors were used: pET22b (Novagen, Madison, WI, USA), pQE31 (Qiagen, Hilden, Germany), pTZ19 (Promega, Madison, WI, USA). The cell strains NovaBlue, BL21(DE3) and AD494 were from Novagen (USA). Sodium lauroyl sarcosinate and β-octylglucoside were from Fluka (Switzerland). αBgt was from Sigma (Deisenhofen, Germany). [125I]αBgt was prepared according to [24] using [125I]NaI (specific activity 2000 Ci· mmol–1) from Isotop (St Petersburg, Russia), or the labeled neurotoxin was purchased from Amersham International (Amersham, Bucks, UK). AChR-enriched membranes were prepared as described in reference [25] from the electric organ of T. californica supplied by Winkler Enterprises (San Pedro, CA, USA). Oligonucleotides were synthesized by Dr N. Bystrov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry).

Cloning of cDNA fragments of the T. californicaα-subunit and their expression in E. coli cells

To prepare the cDNA fragment coding for amino acid residues 1–209 of the α-subunit, two primers complementary to the nucleotide sequences 1–15 and 613–627 of the T. californicaα-subunit cDNA were synthesized. At the 5′-end, the primers had restriction sites for BamHI and HindIII, respectively. The primers were used to obtain a cDNA fragment from the T. californicaα-subunit cDNA (provided by Dr D. Beeson, Oxford), and the product was subcloned into SmaI-digested vector pTZ19. The target fragment was cloned into pET22b and/or pQE31 vectors using the BamHI-HindIII sites. The recombinant plasmids were transfected into NovaBlue cells. The plasmids (designated α1-209pET and α1-209pQE) were then used to transfect the E. coli strain BL21(DE3) or AD494, respectively. After induction with isopropyl β-d-thiogalactopyranoside (IPTG), the efficiency of expression was examined, and one clone in which the target protein accounted for over 30% of the total cell protein was selected. BL21(DE3) cells were lyzed in 6 m guanidinium chloride, and centrifuged cell lysate was applied on to a Ni2+-NTA-agarose (Novagen) equilibrated in 6 m guanidinium hydrochloride, 50 mm Tris/HCl (pH 8.0). The column was washed with 8 m urea in 50 mm Tris/HCl (pH 8.0), and then the product was eluted by 1 m imidazole in 8 m urea.

For α1-209pET protein, urea and imidazole were removed by gel chromatography on a Sephadex G-25 column equilibrated with 150 mm NaCl, 10 mm phosphate buffer pH 7.6, 1% β-octylglucoside.

When the pQE31 vector and AD494 cells were used, after induction with IPTG the cells were harvested by centrifugation, suspended (1 g of wet cells in 50 mL) in cold 10 mm Tris/HCl buffer pH 8.0 containing 1 mm EDTA and treated with lysozyme (final concentration 0.1 mg·mL–1). The urea was added to give the concentration of 8 m, the mixture was sonicated (3 × 30 s), centrifuged (15 min, 10 000 g), and after addition of MgCl2 (final concentration 10 mm) the supernatant was applied on to an Ni2+-NTA column equilibrated with 8 m urea. The product was eluted with 1 m imidazole in 8 m urea, and then subjected to stepwise dialysis. Each step lasts for 2 h and include dialysis against: (a) 50 mm Tris/HCl (pH 8.0) with 6 m urea; (b) the same buffer with 3 m urea; (c) the same buffer with 1.5 m urea; and (d) the same buffer without urea. After centrifugation of precipitate the yield was 15–20% (calculated with respect to 50–60 mg of the target protein in 1 L of the culture medium).

The cDNA fragment coding for amino acids 125–265 of the α-subunit AChR of the T. californica was similarly prepared using the primers corresponding to the nucleotide sequences 373–387 and 781–795. It was cloned into pET22b vector at the BamHI and HindIII sites and expressed in BL21(DE3) cells. In this case, the protein was isolated from inclusion bodies using lysozyme (1 mg·mL–1) and sonication. After centrifugation, the pellet was first washed with 25 mm Tris/HCl pH 8.5, containing 1% Triton X-100, 10 mm EDTA, 0.4 mm PMSF, and then with the same buffer without Triton X-100. Finally, the pellet was solubilized in 20 mm Tris/HCl buffer, pH 7.9, containing 0.5% sodium lauroyl sarcosinate, 5 mm imidazole, 0.5 m NaCl, and applied on to a Ni2+-NTA-agarose column. The column was first washed with the same buffer containing 100 mm imidazole, than the product was eluted with the buffer containing 1 m imidazole.

Sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). Electrophoresis was performed according to Laemmli [26] with 1.5-mm gels on a Midget electrophoresis unit (LKB, Bromma, Sweden). Gels were stained with Coomassie Brilliant Blue R-250 or BioRad Silver Stain (Bio-Rad, Richmond, CA, USA).

Analytical HPLC

Reverse-phase HPLC separations were made on a System Gold chromatograph (Beckman, Fullerton, USA) using Nucleosil 300–7 Protein PR or Nucleosil 300–5 C4 MPN columns (both 4 × 125 mm, Macherey-Nagel, Düren, Germany). Size-exclusion HPLC was performed on Spherogel TSK 4000SW (13 µm, Beckman) or UltroPac TSK-G 3000SW (LKB, Bromma, Sweden) columns (both 7.5 × 600 mm).

Protein concentrations

These were determined by three methods: (a) protein-dye binding method of Bradford [27]; (b) from absorbance at 280 nm using ε280 of 60 868, 62 348, and 37 228 m–1·cm–1 for α1-209pQE, α1-209pET and α125-265pET, respectively, as calculated for these proteins from their amino acid composition [28]; and (c) from the difference of absorbances at 235 and 280 nm [29]. The two methods based on ultraviolet absorbance measurements gave close values and were used to estimate the protein in the purified preparations used for CD analysis and for binding studies.

Edman degradation

This was performed on a Model 473A protein sequencer (Applied Biosystems).

MALDI mass spectrometry

This was carried out on a Reflex MALDI-TOF mass spectrometer (Bruker, Bremen, Germany). The protein samples obtained after chromatography on a Ni2+-NTA column were diluted 100–200-fold with 50% aqueous AcCN containing 0.1% trifluoroacetic acid. The same system was used for analysis of lyophilized fractions after HPLC. The spectra were obtained with α-cyano-4-hydroxycinnamic acid (Sigma) as a matrix.

CD spectra

These were measured at 22 °C using a Jasco Model J-500C spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan) in semiautomatic slit adjustment mode. The scan speed was set at 5 nm·min–1, the response time at 4 s, and the scan ranges were from 190 to 240 nm. The instrument was calibrated with (+)-10-camphorsulfonic acid. Optical activity was expressed as mean residue ellipticity, [Θ], in degrees·cm2·dmol–1, based on mean residue weight of 118 for α1-209pQE and 117 for α1-209pET. Protein concentration varied from 0.2 to 1.5 mg·mL–1, and the cell path length was either 0.1 or 0.5 mm. All samples were optically homogeneous. Secondary structure was calculated from the CD data using the contin program [30,31].

Interaction of AChR and its fragments with [125I]αBgt, low-molecular-weight antagonists, and monoclonal antibodies (mAb)

Binding of [125I]αBgt after SDS/PAGE was demonstrated by transfer to nitrocellulose and overlaying with solutions of [125I]αBgt (100 nm) for 1 h at room temperature. To measure binding in solution, either aqueous solutions of the proteins were used or β-octylglucoside was added to a final concentration of 1%. After incubation with [125I]αBgt, the receptor–domain toxin complex was separated from unbound [125I]αBgt by the DE-81 filter disk assay either as originally described [32] or using fast filtration as in Kachalsky et al. [33]. In competition experiments, the proteins were first preincubated with d-tubocurarine or decamethonium bromide for 60 min, and then [125I]αBgt was added. After further incubation for 15 min, the solutions were applied on to DE-81 filters and rapidly washed with 50 mm Tris/HCl (pH 8.0, 5 × 1 mL). To measure binding of monoclonal antibodies, α1-209pQE protein (4 mg·mL–1) was diluted 1 : 200 in Tris/HCl 1% β-octylglucoside and labeled with 4 nm[125I]αBgt. Fifty-microliter aliquots were incubated overnight with 50 µL of 1 : 1000 dilution of each mAb followed by immunoprecipitation with 25 µL sheep antimouse IgG (with 5 µL normal mouse serum to provide carrier IgG). Precipitates were centrifuged and washed briefly before counting on a γ-counter (Packard, Meriden, CT, USA). Results are expressed as fmol precipitated per assay.

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Production and characterization of recombinant polypeptides

Expression in E. coli usually faces a problem of obtaining the recombinant proteins as water-soluble and correctly folded products. Quite often, recombinant proteins are accumulated in E. coli within the so-called inclusion bodies, which entails their poor solubility. One way to achieve correct folding in E. coli is to use vectors that code for signal sequences of proteins that are efficiently secreted into E. coli periplasm: the leader sequence should be selectively cleaved off during translocation through the ER membrane while the protein wanted would acquire the correct folding and disulfide bond formation. Another possibility is to use cells which facilitate formation of correct disulfide bridges in vivo. We tried these two approaches in order to obtain the N-terminal domain 1–209 of the T. californicaα-subunit, using namely (a) pET22b vector containing the leader sequence of the pelB protein, and (b) pQE31 vector and expression in the AD494 cells. Both of them code for a (His)6 tag, which is situated close to the C-terminus of polypeptides produced in pET22b vector, and at the N-terminus in the pQE31 constructs. We also used the pET22b vector in an attempt to obtain a polypeptide comprising residues 125–265 of the α-subunit, that is roughly a half of the N-terminal extracellular domain plus the transmembrane sequences M1 and M2.

Although it was anticipated that the protein corresponding to the N-terminal extracellular domain of the AChR α-subunit would be soluble in aqueous media, the α1–209 pET polypeptide was found mainly in the inclusion bodies. Not surprisingly, the same was found with the α125–265 pET polypeptide that comprises two transmembrane fragments. However, the protein expressed in the AD494 cells with the aid of pQE31 vector, designated α1-209pQE, had much better solubility in aqueous media: about 15–20% of the total expressed protein was found in the supernatant, not associated with the inclusion bodies. However, for practical reasons, a high concentration of urea or guanidinium chloride was used at the first stage to solubilize all recombinant polypeptides (α1-209pET, α125-265pET and α1-209pQE).

As can be seen from Fig. 1A, the (His)6 tag allowed us to isolate in one step the products α1-209pET and α1-209pQE, which appeared to be electrophoretically almost homogeneous (when analyzed in reducing or nonreducing conditions, cf. lanes 3, 4 and 6 in Fig 1A). The expression of the 125–265 fragment was considerably weaker than that of the 1–209 fragment. In addition to the main band of expected molecular mass (≈ 19 kDa), a band of higher molecular mass (≈ 33 kDa) could also be detected by Coomassie staining (Fig. 1B). Autoradiography after overlay of nitrocellulose blots (Fig. 1C), demonstrated that [125I]αBgt bound to a single band both in the case of α1-209pET and α1-209pQE fragments. With the α125-265pET protein it bound to the main band and, although less efficiently, to the band of ≈ 33 kDa.

image

Figure 1. SDS/PAGE analysis of the Torpedo AChR α-subunit domains expressed in E. coli. (A) (1) Standards; (2) cell lysate, (3 and 4) purified α1-209pQE protein under reducing and nonreducing conditions, respectively; 5- and 6-cell lysate and purified α1-209pET protein. (B) (1) Standards, (2) α125–265pET polypeptide eluted from the Ni2+-NTA column, (3) fraction obtained on subsequent purification of this protein by reverse-phase HPLC (see Fig. 2). (C) Autoradiography of the α1-209pQE (1) and α125–265pET (2) proteins after SDS/PAGE and incubation with [125I]αBgt.

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Figure 2 shows the results of the reverse-phase HPLC analysis of the recombinant proteins. In the case of the α1-209pET and α1-209pQE proteins, obtained after removal of urea and spontaneous air oxidation of cysteine SH-groups, one relatively broad peak is observed. The broadness of peaks (Fig. 2A,B) may be explained by the presence of different forms of a similar molecular mass (as follows from the dominance of one band on SDS/PAGE) that have similar hydrophobic properties. Noteworthy, when the α1-209pQE was applied on to a reverse-phase HPLC column immediately after elution from the Ni2+-NTA-agarose column or after reduction in denaturing conditions, the main peak was considerably narrower (see a dotted profile in Fig. 2B).

image

Figure 2. Reverse-phase HPLC of the Torpedo AChR α-subunit domains expressed in E. coli. (A) α1-209pET protein, Nucleosil 300–5 C4 MPN column; the sample volume is 25 µL (4.5 mg·mL–1) in 50 mm Tris/HCl buffer, pH 8.0, 100 µm EDTA. The column was operated in gradient of solvent B [0.1% TFA in AcCN/H2O mixture (4 : 1)] concentration in solvent A (0.1% TFA in H2O), flow rate 0.5 mL·min–1. (B) α1-209pQE protein, a Nucleosil 300-7 Protein RP column; protein samples of 20 µL (4.6 mg·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 6 m urea and 1 m imidazole immediately after elution from Ni2+-NTA-agarose column (dotted line) and of 100 µL (1.8 mg·mL–1) in 10 mm sodium phosphate buffer pH 7.3, 0.4 m urea, 1 mm EDTA after incubation in this solution for several weeks (solid line); the absorbances of the former chromatogram were multiplied by 1.32 for better comparison. The column was operated in gradient of solvent B [0.1% TFA in AcCN/H2O mixture (19 : 1)] concentration in solvent A (0.1% TFA in H2O), flow rate 0.5 mL·min–1. (C) α125–265pET protein, a Nucleosil 300–7 Protein RP column; protein sample is 820 µL (0.3 mg·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 7 m urea, and 10% SDS. The column was operated in gradient of solvent B (0.1% TFA in AcCN) concentration in solvent A (0.1% TFA in H2O) at flow rate of 0.5 mL·min–1.

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We also used reverse-phase HPLC to monitor various reoxidation-refolding procedures. When disulfides in the α1-209pQE or α1-209pET proteins were reduced in denaturing conditions followed by a decrease in the denaturant concentration by dialysis or gel filtration, over 80% of the protein precipitated. The protein remaining in solution after reoxidation, gave a reverse-phase HPLC profile very close to that of the starting (spontaneously oxidized) protein (data not shown).

In another experiment, the α1-209pQE protein was treated with protein-disulfide isomerase in the presence of 0.5–1 mm cysteine or reduced glutathione. Again, such a treatment brought about only small changes in the reverse-phase HPLC characteristics (not shown). As these approaches did not change dramatically the properties of the recombinant proteins, binding studies and CD analysis (see below) were performed with the proteins obtained after spontaneous oxidation.

The reverse-phase HPLC profile for the α125-265pET protein shows the presence of several components (Fig. 2C). SDS/PAGE analysis of the main peak (marked with a bar on the chromatogram) demonstrates (see Fig. 1B) the enrichment in component with Mr≈ 19 kDa, although forms of higher molecular mass (≈ 33 and 43 kDa) could also be detected in some experiments.

The molecular masses of the expressed AChR α-subunit fragments were determined with the aid of MALDI mass spectrometry (Fig. 3). The spectrum of the α1-209pET protein confirms its homogeneity: all the observed peaks appear to be derived from one peak of 29568 (M + H+), peaks 14791, and 9869 having two and three positive charges, respectively, while a low-intensity 59158 peak indicates the presence of a dimer of the α1-209pET protein. Although the molecular ion with a maximum intensity at 21154 m/z and a respective mass with two positive charges (10581) are observed for the α125-265pET protein (Fig. 3B), the broadness of the signals and the presence of other masses reveal the heterogeneity of the preparation. As can be seen from Table 1, the measured molecular masses of α1-209pET and α125–265pET proteins exceed by about 2200 the calculated ones [on account of the (His)6 tag and polylinker fragments]. Edman degradation of both proteins revealed the same sequence MKYLLPTAA corresponding to the leader peptide of the pelB protein. Taking into account these additional sequences (see Table 1), the calculated molecular masses for the α1-209pET and α125-265pET proteins are 29380 and 21106, respectively. These values are in reasonable agreement with the experimentally determined masses (Table 1). In summary, MALDI mass spectrometry and Edman degradation show that the presence of the pelB leader sequence in the pET constructs failed to ensure the secretion of the proteins, and resulted in an undesirable lengthening of the expressed sequences.

image

Figure 3. MALDI mass spectra of the Torpedo AChR α-subunit domains. (A–C) α1-209pET, α125–265pET and α1-209pQE proteins, respectively.

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Table 1. Molecular masses of expressed proteins.
  Molecular mass
DesignationExpected sequenceCalculatedFound
α1-209pETMDIGINSDP-(1–209AChR)-KLAAALA-H627 374 
MKYLLPTAAAGLLLLAAQPAMA-MDIGINSDP -(1–209AChR)-KLAAALE-H629 38029 568
α125–265pETMDIGINSDP-(125–265AChR)-KLAAALE-H618 895 
MKYLLPTAAAGLLLLAAQPAMA-MDIGINSDP -(125–265AChR)-KLAAALE-H621 10621 154
α1-209pQEMRSP H6-TDP-(1–209 AChR)-KLN26 61726 651

The molecular mass of the α1-209pQE protein, determined by MALDI mass spectrometry (Fig. 3C), was in satisfactory agreement with the calculated one (Table 1), and Edman degradation showed the presence of residues M-R-X-X-H6-T-D-P (from the vector) followed by the expected N-terminal sequence (SEHE) of the T. californica AChR α-subunit. Again, MALDI spectra provided the evidence for the chemical homogeneity of the α1-209pQE protein: there are no other peaks except the molecular ion (26651), its double-charged fragment (13335), and a low-intensity peak (53331), probably corresponding to a dimer.

As MALDI spectra provided evidence for the presence of a dimer, in addition to a monomer (see Fig. 3), we examined the α1-209pQE protein by gel-permeation chromatography. Figure 4 shows the predominance of the monomer (marked with an arrow at 26 kDa), whereas peaks that could be ascribed to oligomers are also present. These oligomers do not contain intramolecular disulfide bridges, since, as seen from Fig. 1A, the SDS/PAGE patterns obtained under reducing and nonreducing conditions do not differ.

image

Figure 4. Size-exclusion HPLC of the α1-209pQE protein on a Spherogel TSK 4000SW column. A sample volume is 25 µL (4 mg protein·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 100 mm LiCl, 100 µm EDTA. Isocratic elution was performed at a flow rate of 0.5 mL·min–1 using the same buffer.

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Binding of [125I]αBgt, low-molecular mass antagonists and monoclonal antibodies to α1-209pET and α1-209pQE proteins

All expressed α-subunit fragments, as mentioned above, were found to bind [125I]αBgt on blots, but it was important to investigate the efficacy of binding in solution. αBgt binding to α1-209pET and α1-209pQE proteins (Fig. 5) was performed in aqueous solution of 1% β-octylglucoside. These conditions we have previously shown [34] to allow investigation of binding to isolated α-subunits of T. californica AChR after purification by SDS/PAGE and HPLC. KD values for the α1-209pET and α1-209pQE proteins were 141.4 ± 10.6 and 127.0 ± 11.3 nm, respectively, being only about fourfold higher than the value (30 nm) previously found for the isolated α-subunits under the same conditions [34]. Bmax values were 9.5 ± 0.2 and 10.3 ± 0.2 nmol·mg–1, compared with theoretical maxima of 34 and 38 nmol·mg–1, respectively. Apparently, the pelB sequence in the α1-209pET protein, and the position of the His6 tag either at the C-terminus or N-terminus exerted no significant effects on the binding parameters. The KD values obtained are in the range of values reported for partially renatured α-subunit from Torpedo, and for α-subunit N-terminal fragments expressed as fusion proteins (see references [20–22,35,36]). However, with most of the expressed α-subunit fragments described previously, the fraction of the protein that bound [125I]αBgt, as calculated from the Bmax was a few percent, whereas in our case the values are 27–28%.

image

Figure 5. Specific binding of the [125I]αBgt to the α1-209pQE (closed circles) and α1-209pET (open circles) proteins. The total concentration of both proteins dissolved in 50 mm Tris/HCl buffer pH 8.0, 1% β-octylglucoside, was 400 nm. Nonspecific binding was determined in the presence of αBgt taken in 100-fold excess with respect to each concentration of the radioactive ligand. Each point is an average of triplicate measurements. KD and Bmax were calculated by means of Enzfitter (Elsevier Biosoft). Inset: Scatchard plot.

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The fact that even this value is far from 100% should not be discouraging. Noteworthy, even the whole α-subunit had no more than 50% of the theoretical αBgt-binding capacity, unless associated with the γ- and δ-subunits [37]. The activity of the SDS/PAGE-isolated α-subunit measured, in terms of αBgt-binding sites, in the presence of various detergents, was reported by different authors to span the range of 35–95% [35,36,38]. In our hands, SDS/PAGE of the Torpedo membranes followed by reverse-phase HPLC and transfer to β-octylglucoside gave an α-subunit preparation that bound αBgt with KD≈ 28 nm and Bmax 0.5 nmol·mg–1 (≈ 2.5% activity) [34,39].

The lower affinity of that preparation as compared with native AChR was apparently due to incomplete renaturation after SDS/PAGE/HPLC, as well as to a loss of interactions with the neighboring γ- and δ-subunits. It is not clear which other factors further reduce the affinity of the expressed α-subunit domain 1–209, as compared with that of the complete α-subunit. One possibility is that, unlike the subunits obtained from purified Torpedo AChR, the proteins expressed in E. coli are not glycosylated. However, the data on the role of carbohydrates in the interaction of Torpedo AChR with αBgt are conflicting [40–42].

We attempted to increase the activity of the 1–209 proteins with the aid of affinity chromatography with immobilized α-neurotoxins. In fact, this step previously allowed Fraenkel et al. [43] to increase the toxin-binding capacity of the fusion protein encompassing the α-subunit fragment 184–200 from 3.3 to 16.5% and to use it for NMR analysis of ligand binding. Indeed, between 10 and 60% of the total protein in different preparations of the α1-209pET and α1-209pQE could be bound to the Sepharose columns with the attached neurotoxin II Naja naja oxiana or α-cobratoxin Naja naja siamensis. However, high concentrations of carbachol required to elute the specifically bound protein were found to precipitate it from aqueous solutions. Elution could be done by 1 m carbachol in 1% CHAPS. However, the binding parameters of the proteins thus eluted did not improve considerably as compared with the starting preparation.

To test the ability of the expressed proteins to interact with low molecular weight antagonists whose binding is more sensitive to the structure of the ligand binding site, we examined the interaction of [125I]αBgt with α1-209pQE in the presence of varying concentrations of d-tubocurarine and decamethonium (Fig. 6). These studies were performed in purely aqueous solution: the absence of β-octylglucoside did not affect the Bmax value (9.7 ± 0.3 nmol·mg–1), while the affinity was found to be about twofold higher (KD 75.0 ± 6.8 nm). The IC50 values determined, 200 µm for d-tubocurarine and 8 mm for decamethonium, are very close to those obtained earlier [36,44] for isolated α-subunits. It means that the expressed protein, retains, at least partially, the structure of the ligand-binding site.

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Figure 6. Inhibition of [125I]αBgt specific binding to α1-209pQE by d-tubocurarine (squares) and decamethonium bromide (circles). Concentration of the protein in 50 mm Tris/HCl, pH 8.0 was 3 µm. Nonspecific binding was determined as indicated in the legend to Fig. 5. Each point is an average of duplicate measurements. C = concentration of the competitor. IC50 were calculated by means of Origin 2.94 (Microcal Software, Inc.).

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One characteristic of the intact AChR is binding of conformation-dependent antibodies. Of the mAbs previously described by Whiting et al. [45] raised against native Torpedo AChR, only two, M2D11 and M4D11, bound appreciably to the α-subunit on Western blots of purified Torpedo AChR [46], and M4D11 bound to both α1-209pQE and α1-209pET, although M2D11 did not (Fig. 7). However, both mAb M2C2, and to a lesser extent M5B5, bound to [125I]αBgt-labeled α1-209pQE and α1-209pET. Binding of these mAbs is conformation-dependent and directed at the αBgt-binding site that exhibits lower affinity for d-tubocurarine and other competitive ligands [45]; they inhibit [125I]αBgt binding to one site on the AChR, and do not precipitate Torpedo AChR that is fully saturated with [125I]αBgt. Yet, both α1-209pET and α1-209pQE apparently bind to a site that is separate from the αBgt-binding site, because they immunoprecipitate [125I]αBgt-labeled proteins (Fig. 7) and do not inhibit [125I]αBgt binding (data not shown). Thus, these proteins express at least some of the highly conformational antigenic sites that were previously described on Torpedo AChR [45], but they may be arranged slightly differently, perhaps because of the lack of interaction with the adjacent subunits that clearly influence the binding sites [2,3].

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Figure 7. Binding of monoclonal antibodies to the expressed proteins. Immunoprecipitation of [125I]αBgt-prelabeled α1-209pET and α1-209pQE proteins by monoc1ona1antibodies against AChR [45,46], compared with precipitation of T. californica[125I]αBgt-prelabeled AChR. Non-site mAbs and anti-site mAbs refer to antibodies that do not or do compete with [125I]αBgt for its binding site (the one with lower affinity for d-tubocurarine [45,46]) on Torpedo AChR. The results are expressed as fmol of binding sites precipitated per assay and are the mean ± SD of at least two determinations.

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As the expressed proteins interact with a specific neurotoxin (αBgt), with low-molecular mass antagonists (d-tubocurarine and decamethonium) and with Torpedo AChR-specific antibodies (that do not cross-react well with AChR from other species [45]), they should be useful models for the examination of the spatial structure of the AChR extracellular domain. As a first step along these lines, we measured CD spectra of the expressed proteins (see Fig. 8 and Table 2).

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Figure 8. CD spectra of the Torpedo AChR α-subunit domains expressed in E. coli. (1 and 2) α1-209pET protein (0.7 mg·mL–1) and α1-209pQE protein (1.0 mg·mL–1), respectively, in 50 mm Tris/HCl buffer (pH 8.0) with 100 µm EDTA [in order to measure the spectra of the α1-209pET protein in aqueous solution, the starting solution of the protein (3.5 mg·mL−1) containing 1% β-octylglucoside was first diluted fivefold, and then dialyzed extensively against the indicated buffer], (3) α1-209pQE protein (0.5 mg·mL–1) in 10 mm sodium phosphate buffer pH 7.3, 0.2% SDS and 500 µm EDTA, and (4) α1-209pQE protein (0.8 mg·mL–1) in 50% aqueous TFE.

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Table 2. Secondary structure of the Torpedo AChR α-subunit domains expressed in E. coli as calculated from the CD spectra of the respective proteins in different conditions.
ProteinsSolventsα-helix (%)β-sheet (%)β-turn (%)Random coil
α1-209pET50 mm Tris/HCl, pH 8.03556010
α1-209pQE50 mm Tris/HCl, pH 8.03250810
α1-209pET10 mm phosphate Na, pH 7.3, 0.2% SDS36322011
α1-209pQE50% TFE5130613

CD spectra of α1-209pET and α1-209pQE proteins

The far-ultraviolet CD spectra of α1-209pET and α1-209pQE proteins in Tris/HCl buffer (Fig. 8, curves 1 and 2) were characterized by a positive Cotton effect in the 190–200 nm region, and a negative one in the 200–240 nm region. Such a spectrum suggests a major contribution from β-sheet structure. However, quite a high dichroism intensity over a relatively wide region around 215 nm is indicative of the presence of bands at 208 and 222 nm (π–π* and n –π* transitions, respectively), characteristic of α-helices [47]. The calculation by the CONTIN program [30,31] showed that 32% of amino acid residues in α1-209pQE protein have an α-helical conformation, 50% form a β-pleated sheet, while β-turns and random coil account for 8 and 10%, respectively (Table 2). The α1-209pET protein has a very similar structure: there is only a weak (3–6%) increase in the α-helix and β-structure content (at the expense of β-turns). Apparently, neither the pelB leader sequence, nor the location of the His6 tag closer to the N- or C-termini, greatly change the secondary structure. We also found that the presence of 0.5–1% CHAPS or β-octylglucoside as well as the treatment of proteins by the protein disulfide isomerase did not perturb the CD spectra (data not shown). Adding 0.2% SDS, an anionic surfactant, entails significant changes of secondary structure, with increase in the α-helix and β-turn contents and a concomitant decrease in β-structure (curve 3 in Fig. 8, Table 2).The most visible changes in the CD spectra were caused by 50% trifluoroethanol (TFE), which increased the helicity to 50% (Fig. 8). This is not surprising as the α-helix promoting activity of TFE is well known [48]. Thus, the CD spectra show that the expressed N-terminal domain in aqueous solution has a defined secondary structure, which can be perturbed by changing the environment upon adding SDS or TFE.

As low concentrations (0.02%) of SDS were reported to increase the affinity of the isolated α-subunit [36] and of synthetic peptides [49] for αBgt, we checked possible effects of 0.02% SDS on the CD spectra of the 1–209 proteins. No marked changes, as compared with the spectra taken in aqueous buffers, were observed (data not shown), indicating that possible alterations in the affinity for α-neurotoxins are not necessarily associated with reorganizations of the secondary structure. In fact, we could not detect marked effects of αBgt or d-tubocurarine (antagonists) or of acetylcholine and carbachol (agonists) on the CD spectra of the α1-209pET or α1-209pQE proteins either in the absence, or in the presence of 0.02% SDS. As the capacity of d-tubocurarine and decamethonium bromide to interact with the α1-209pQE protein was disclosed (via their competition with [125I]αBgt), it means that their binding is not accompanied by gross changes of the secondary structure (small changes would not have been visible because of the ≈ 25% activity of our preparations).

Secondary structure and functional properties of various AChR domains

It is interesting to compare the results obtained with previous data on the secondary structure of the whole AChR or its domains. From Fig. 1 in [50], based on the analysis of the sequences of all T. californica AChR subunits, we deduced the following secondary structure composition for the 1–209 region: α-helix 30%, β-structure 45%, β-turns 19%, and random coil 6%. These values are quite close to those determined by us experimentally, especially for the α1-209pQE protein in aqueous solution.

A recent attempt to envisage the spatial structure of the Torpedo AChR extracellular domain, involved modeling of the α-subunit sequence 31–200 according to the X-ray structures of plastocyanin and pseudoazurin with which it shares about 30% homology [51]. The model postulated the predominance of β-structure (nine β-strands) and the presence of three α-helices, which in total would give 19% α-helical structure.

As far as direct experimental estimation of the secondary structure is concerned, CD and FTIR data are available for the whole Torpedo receptor, either free or in a liganded form [52–54]. Information on the secondary structure of the intramembrane domain was obtained by simultaneous chopping off the extracellular and intracellular domains by proteinase K, and subsequent FTIR analysis of the residual membranes [55]. Our data are direct determinations of the secondary structure of polypeptides that represent the extracellular N-terminal domain of the Torpedo AChR α-subunit. The predominance of β-structure disclosed by CD spectra may explain the propensity of the expressed α1–209 fragment to form aggregates, but it can also be an important element in the assembly of the α-subunit and the other subunits into the intact pentaoligomeric AChR complex. On the other hand, the 32% percentage of α-helices is sufficient to form the three helices whose presence in the extracellular domain of the Torpedoα-subunits was inferred from cryo-electron microscopy data [16].

After our results were reported in an abstract [56], Lester and collaborators published the expression in the Chinese hamster ovary cells of the protein comprising residues 1–210 of the mouse muscle AChR α-subunit [23]. The protein obtained is glycosylated, does not have a His6 tag and is reported to have a very high affinity for αBgt, although the binding capacity was only about 40%. The secondary structure calculated from the CD spectrum of this protein (which has a 73% sequence identity to the T. californicaα-subunit in the 1–209 region) was 12% α-helix, 51% β-structure, 18% β-turns, and 20% random coil. The common feature for the N-terminal domains of the α-subunits from these two different species, expressed either in eukaryotic or prokaryotic systems, is the prevalence of β-structure (50–65%) and the presence of appreciable amounts of α-helices (12–32%).

Thus, one can see that the secondary structure determined for the 1–209 proteins expressed in E. coli is in general agreement with the theoretical predictions and the experimental results for a related protein [23]. As the 1–209 protein was found to bind αBgt and low-molecular mass antagonists, and also preserved some of the conformational antigenic sites (see above), we believe that this protein is similar to the respective domain of the intact receptor not only with respect to its secondary structure, but also with certain elements of the topology. The situation is reminiscent of the protein state known as ‘molten globule’, which has essential features of the native secondary structure and of the native overall architecture, but lacks the native tertiary structure characterized by well-packed side chains [57].

Other recent publications demonstrate that heterologously expressed extramembrane fragments of the oligomeric ligand-gated or voltage-gated ion channels are adequate models for elucidating the structure of the respective domains of the intact molecules [58–61]. Interestingly, the extramembrane fragment of the Shaker potassium channel expressed in E. coli gave exclusively tetramers (that are also characteristic for intact channels) whose three-dimensional structure was solved by X-ray crystallography [61]. Expression in Xenopus oocytes of the N-terminal domain of the α7 subunit of the pentaoligomeric α7 AChR was also found to result in oligomers, most probably pentamers [60].

In view of these results we believe that expression of the N-terminal domain of Torpedo AChR α-subunit in E. coli, reported in the present communication provides large amounts of a protein which is, indeed, an appropriate model for elucidating important aspects of the AChR structural organization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

The authors are grateful to Dr D. Beeson (Institute of Molecular Medicine, Oxford) for providing a cDNA clone of the T. californica AChR α-subunit, to Drs C. Weise and P. Franke (Free University of Berlin) for help in sequencing and mass spectrometry analyses. Fruitful discussions with Dr C. Methfessel (Bayer AG, Leverkusen) are highly appreciated. This work was supported by a cooperation grant from the German Federal Ministry for Research and Technologies (BMBF) and the Russian Ministry of Science. Financial support by the Russian Foundation for Basic Research (grant 96-04-50375), the Deutsche Forschungsgemeinschaft (Sfb312), the Fonds der Chemischen Industrie and by Bayer AG (Leverkusen) is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References
Footnotes
  1. Enzymes: lysozyme (EC 3.2.1.17); protein-disulfide isomerase (EC 5.3.4.1); restriction endonuclease BamHI; restriction endonuclease HindIII; restriction endonuclease SmaI