Label Transfer: A Key Step in Mapping Short Î±

Bioconjugate Chem. 2006, 17, 1482−1491

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Photocrosslinking/Label Transfer: A Key Step in Mapping Short r-Neurotoxin Binding Site on Nicotinic Acetylcholine Receptor Pascal Kessler,* Robert Thai, Fabrice Beau, Jean-Luc Tarride, and Andre´ Meńez CEA, DSV, DIEP, Gif-sur-Yvette, F-91191, France. Received June 19, 2006; Revised Manuscript Received August 23, 2006

We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific R-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin-nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the R subunit (59%), followed by the γ subunit (28%), with the δ subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. “In-gel” CNBr cleavage of the labeled R subunit followed by Edman degradation of the purified peptides showed that RTyr190 and RTyr198 were the most labeled residues, with a less important labeling on RCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.

INTRODUCTION With relation to the discovery of soluble acetylcholine binding proteins (AChBP’s1) in various snails, the recent past has been quite rich in structure descriptions of complexes between nicotinic ligands and the surrogate of the acetylcholine receptor. These data brought new insights on the interactions between agonists/antagonists and their intrinsic acetylcholine protein target. X-ray crystallography of complexes between AChBP and agonists, such as nicotine, carbamylcholine (1), epibatidine, or lobeline (2) showed that the critical C loop, classically harbored by R-subunits on nAChRs, wraps these ligands, occluding the binding site. In contrast to these data, structures of complexes with peptidic antagonists, such as R-conotoxin ImI (2), R-conotoxin PnIA (3), or R-cobratoxin (4) showed a more or less extended C loop, its position depending on the steric hindrance of the antagonist. Three finger curaremimetic snake R-neurotoxins can be divided into long-chain and short-chain toxins. R-Bungarotoxin and R-cobratoxin belong to the first group, which binds with high affinity to the neuronal R7 receptor and to AChBP. Crystals and X-ray data of a complex between AChBP and R-cobratoxin have been obtained recently by Bourne and colleagues (4), revealing an unpredicted conformation of the toxin-bound AChBP, especially of loop C on the R subunit. NmmI, erabutoxin-a, NTII, and the R-neurotoxin of Naja nigricollis belong to the short-chain group, which is specific to the muscle-type nAChR. This family of toxins do not bind with * Address correspondence to: Pascal Kessler, CEA, DSV, DIEP, Baˆtiment 152, courrier Nο 24, Gif-sur-Yvette, F-91191, France. Tel. 33-1-69-08-40-74; Fax. 33-1-69-08-90-71; E-mail: [email protected]. 1 The abbreviations used are as follows: nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine binding protein; SDS, sodium dodecyl sulphate; DTT, dithiothreitol; TCEP, Tris-carboxyethylphosphine; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; dtb, desthiobiotin; dtb-R33C, Naja nigricollis N-terminal-desthiobiotinylated R-neurotoxin cysteine analogue; dtb-R33C-ArN2+, Naja nigricollis N-terminal-desthiobiotinylated R-neurotoxin cysteine analogue harboring an aryldiazonium salt at position 33; R33C-DABD, R-neurotoxin cysteine analogue harboring a diaminobenzenediazonium salt at position 33.

high affinity to AChBP (4, 5), and no structure of a complex with AChBP has ever been published. However, by checking the ability of making ternary complexes between nAChR, streptavidin, and biotinylated analogues of Naja nigricollis R-neurotoxin, it was concluded that short and long curaremimetic toxins share a similar overall topology of interaction when bound to nicotinic receptors (6). Moreover, by comparing the structure of the R-cobratoxin-AChBP complex to data obtained by double mutant cycle analysis of a muscle nAChR and the short-chain R-neurotoxin NmmI (7, 8), Bourne and colleagues suggested that NmmI binds at the subunit interface of the muscle-type nAChR in a somewhat different orientation than R-cobratoxin, with Arg33 acting as a pivot. More recently, an in silico study suggested that the way of entry into the nAChR binding pocket of the second loop of NTII, another short-chain R-neurotoxin, differs from that of R-cobratoxin (9). In order to test these proposals, we mapped the binding area of the tip of the second loop of a short-chain R-neurotoxin on the acetylcholine receptor by photoaffinity labeling. Unfortunately, with such ligands, this technique almost never led to the characterization of the labeled residues on the targeted protein, due essentially to the noncleavable nature of the covalent bond linking the photoprobe to the toxin. Actually, a labeled residue could only be characterized once (10), when the radioactive label was transferred from the ligand to the receptor. However, the 16.5 Å long linker did not lead to a precise mapping of the binding site. To overcome this drawback, we designed here a novel short bifunctional tritiated aryldiazonium photoprobe. This paper describes the synthesis of the photoprobe followed by its coupling, through a cleavable disulfide bond, to an engineered cysteinyl residue at the putative pivotal position 33 of an analogue of a short-chain R-neurotoxin. Photoaffinity experiments, using this new toxin photoprobe in complex with nAChR on Torpedo marmorata membranes, showed a preferential labeling of the R and γ subunits compared to the δ subunit. Edman degradation of the R-subunit-labeled peptides showed that RTyr190, RTyr198, and, to a lesser extent, RCys192 reacted with the photoprobe. Our results reinforce the idea that long and short R-neurotoxins bind to their receptors in a similar manner, at least with the critical Arg33 residue.

10.1021/bc060175j CCC: $33.50 © 2006 American Chemical Society Published on Web 10/19/2006

Short R-Neurotoxin/nAChR Interaction

EXPERIMENTAL PROCEDURES For the sake of clarity, we decided to use erabutoxin numbering for the amino acids of Naja nigricollis R-neurotoxin as we did in previous papers (6, 11, 12). Peptide synthesis was achieved on an Applied Biosystems 433 apparatus. The Fmoc-protected amino acids were obtained from Novabiochem or Advanced Chemtech. All the chemicals were of the highest purity commercially available. Live Torpedo marmorata were purchased from the Station Biologique d’Arcachon (France). Western blots were carried out using Advance-ECL from Amersham Biosciences. Monoclonal antibodies mAb140, mAb155, and mAb165 were a generous gift of Dr. Tzartos (Hellenic Pasteur Institute, Athens). Goat antirat IgG peroxidase F(ab′)2 fragment (H + L) conjugate was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Synthesis of Naja nigricollis R33C toxin analogue was achieved as described earlier (11). Electrospray mass spectrometry was achieved on a Quattro II from Micromass. NMR spectra were recorded on a Bruker Avance 250 MHz. Melting points were taken on a Kofler device. The irradiating device was composed of a 1000 W mercury lamp (Osram), an aperture, and a series of lenses focusing the polychromatic light to a monochromator (Jobin-Yvon), after which a series of lenses focused the monochromatic light to a thermostatic quartz cuvette containing the nAChR. The light intensity was measured with a radiometer IL1700 (International Light, Newburyport, MA). Each experiment involving photoactivatable compounds was done under sodium light, safe for aryldiazonium derivatives. 2-Hydroxymethyl-6-iodo-4-nitrophenol (1). 2-Hydroxy-5nitrobenzoic acid (10.2 g, 55.7 mmol) was selectively reduced with BH3-THF (2.2 equiv) in dry THF (150 mL), at reflux for 1 h. The borates were solvolyzed in dry MeOH, added to destroy the excess of borane. The mixture was evaporated to dryness. The residue was then solubilized again in dry MeOH, evaporated to dryness, and dissolved in a mixture of MeOH/H2O (200 mL/ 50 mL). After treatment with Na2CO3 (1.5 g), ICl (1 equiv) was added. The mixture was stirred for half an hour and then acidified with citric acid. Iodine was reduced with an excess of sodium thiosulphate. The mixture was then concentrated and extracted with EtOAc. The organic phase was washed with water and brine, dried over MgSO4, and purified by silica gel chromatography (gradient: n-hexane 7/EtOAc 3 to n-hexane 5/EtOAc 5), giving orange oil, which was crystallized in CHCl3. The final yield was 14 g (85%) of orange needles, mp 87 °C. 1H NMR (acetone-d ): δ (ppm) 4.97 (s, 2H), 5.58 (s 6 broad, 1H), 8.15 (d, J ) 2.7 Hz, 1H), 8.50 (d, J ) 2.7 Hz, 1H), 10.07 (sbroad, 1H). 13C NMR (acetone-d6): δ (ppm) 61.8, 83.6, 123.0, 127.0, 133.2, 141.1, 160.8. Anal. (C7H6INO4) calcd %: C 28.50, H 2.05, N 4.75, I 43.02. Found %: C 28.74, H 2.06, N 4.72, I 43.11. 1-Iodo-2-methoxy-3-[(4-methoxyphenyl)diphenylmethoxymethyl]-5-nitrobenzene (2). Compound (1) (1.475 g, 5 mmol) was dissolved in pyridine (10 mL) with (4-methoxyphenyl)diphenylmethoxymethyl chloride (2.4 g, 1.5 equiv) and heated to 65 °C for 1.5 h. The mixture was then evaporated, and the residue was dissolved in EtOAc and washed with H2O, a saturated solution of NH4Cl, and a 10% citric acid solution. The organic phase was dried over MgSO4 and evaporated. The residue was taken in DMF (20 mL). Following the addition of NaHCO3 (2.35 g, 28 mmol) and CH3I (2 mL, 32 mmol), the mixture was stirred for 1 day, after which it was concentrated and taken into EtOAc. The organic phase was washed with water and brine, dried over MgSO4, and purified by silica gel chromatography (eluent: n-hexane 95/EtOAc 5), yielding a white solid (2.33 g, 80%), mp 143 °C. 1H NMR (CDCl3): δ (ppm) 3.65 (s, 3H), 3.79 (s, 3H), 4.33 (s, 2H), 6.85 (dd, J ) 6.8 Hz, J ) 2.1 Hz, 2H), 7.24-7.52 (m, 12H), 8.45 (d, J ) 2.7

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Hz, 1H), 8.55 (d, J ) 2.7 Hz, 1H). 13C NMR (CDCl3): δ (ppm) 55.2, 60.7, 61.3, 87.5, 91.0, 113.3, 124.7, 127.2, 128.0, 128.3, 130.3, 133.5, 134.7, 135.0, 143.9, 144.6, 158.8, 162.3. Anal. (C28H24INO5) calcd %: C 57.84, H 4.16, N 2.41, I 21.83. Found %: C 57.77, H 4.77, N 2.48, I 21.98. {3-Iodo-4-methoxy-5-[(4-methoxyphenyl)diphenylmethoxymethyl]phenyl}carbamic Acid tert-Butyl Ester (3). Nitro compound (2) (1.63 g, 2.5 mmol) was selectively reduced with an excess of fresh NaSH, xH2O (8 g) in a mixture of MeOH (50 mL) and THF (20 mL), under reflux for 2 h. After an evaporation step, the product was dissolved in EtOAc, washed with water and brine, and dried over MgSO4. The amine was then taken in THF (10 mL) and protected with ditertiobutyldicarbonate (1.1 g, 2 equiv) at 50 °C, overnight. Finally, it was purified by silica gel chromatography (eluent: n-hexane 9/EtOAc 1), yielding 1.24 g of a white powder (76.5%), mp 201 °C. 1H NMR (acetone-d6): δ (ppm) 1.51 (s, 9H), 3.52 (s, 3H), 3.80 (s, 3H), 4.20 (s, 2H), 6.92 (dd, J ) 6.8 Hz, J ) 2.2 Hz, 2H), 7.267.58 (m, 12H), 7.89 (d, J ) 2.6 Hz, 1H), 8.17 (d, J ) 2.6 Hz, 1H), 8.55 (sbroad, 1H). 13C NMR (acetone-d6): δ (ppm) 28.5, 55.5, 61.4, 61.9, 80.4, 87.8, 91.7, 114.0, 120.2, 127.8, 128.1, 128.7, 129.2, 131.2, 134.1, 136.2, 138.2, 145.5, 153.1, 153.7, 159.8. Anal. (C33H34INO5) calcd %: C 60.83, H 5.26, N 2.15, I 19.48. Found %: C 60.98, H 5.30, N 2.18, I 19.57. (3-Hydroxymethyl-5-iodo-4-methoxyphenyl)carbamic Acid tert-Butyl Ester (4). Compound (3) (1.55 g, 2.4 mmol) was dissolved in THF (50 mL), and the mixture was diluted with H2O (10 mL) and AcOH (50 mL) and refluxed for 3 h. The solvents were evaporated, and the product was purified by silica gel chromatography (gradient: n-hexane 8/EtOAc 2 to n-hexane 7/EtOAc 3), yielding a colorless oil which turned to a white solid (641 mg, 71%), mp 146 °C. 1H NMR (CDCl3): δ (ppm) 1.50 (s, 9H), 3.17 (sbroad, 1H), 3.75 (s, 3H), 4.65 (s, 2H), 6.87 (sbroad, 1H), 7.27 (d, J ) 2.6 Hz, 1H), 7.80 (d, J ) 2.6 Hz, 1H). 13C NMR (CDCl ): δ (ppm) 28.2, 60.7, 61.4, 80.8, 91.5, 119.5, 3 128.4, 135.0, 135.8, 152.7, 152.8. Anal. (C13H18INO4) calcd %: C 41.17, H 4.78, N 3.69, I 33.47. Found %: C 41.27, H 4.87, N 3.71, I 33.59. Thioacetic Acid S-(5-tert-butoxycarbonylamino-3-iodo-2methoxybenzyl) Ester (5). Methanesulfonyl chloride (107 µL, 1.5 equiv) was added to a mixture of compound (4) (350 mg, 0.92 mmol) and Et3N (195 µL, 1.5 equiv) in dry THF (10 mL) and stirred for 15 min. A mixture of thioacetic acid (294 µL, 4 equiv) and Et3N (570 µL, 4 equiv) in EtOAc (3 mL) was added to the mesylate, and the solution was stirred for 1 h, at room temperature. After concentration, the residue was dissolved in EtOAc and washed with water and brine, dried over MgSO4, and purified by silica gel chromatography (eluent: n-hexane 9/EtOAc 1), yielding 345 mg (85.5%) of yellowish oil. 1H NMR (CDCl3): δ (ppm) 1.50 (s, 9H), 2.35 (s, 3H), 3.80 (s, 3H), 4.13 (s, 2H), 6.49 (sbroad, 1H), 7.18 (d, J ) 2.6 Hz, 1H), 7.88 (d, J ) 2.6 Hz, 1H). 13C NMR (CDCl3): δ (ppm) 28.2, 28.5, 30.2, 61.4, 80.7, 91.6, 121.0, 128.6, 131.6, 135.7, 152.5, 153.3, 195.0. Anal. (C15H20INO4S) calcd %: C 41.20, H 4.61, N 3.20, I 29.02, S 7.33. Found %: C 40.47, H 4.56, N 3.10, I 27.75, S 7.34. Thioacetic Acid S-(5-tert-butoxycarbonylamino-2-methoxybenzyl) Ester (6). Iodide (5) (490 mg, 1.12 mmol) was hydrogenated (P(H2) ) 2 bar) for 1 h in EtOAc (10 mL) in the presence of Pd black (1.3 g) and Et3N (700 µL). The mixture was filtered and washed with water and brine, dried over MgSO4, and purified by silica gel chromatography (eluent: n-hexane 9/EtOAc 1), yielding 240 mg (69%) of colorless oil. 1H NMR (CDCl ): δ (ppm) 1.50 (s, 9H), 2.31 (s, 3H), 3.81 (s, 3 3H), 4.09 (s, 2H), 6.40 (sbroad, 1H), 6.77 (d, J ) 8.8 Hz, 1H), 7.19 (d, J ) 2.7 Hz, 1H), 7.34 (dd, J ) 8.8 Hz, J ) 2.7 Hz, 1H). 13C NMR (CDCl3): δ (ppm) 28.2, 28.3, 30.3, 55.8, 80.2, 110.9, 119.4, 121.7, 126.2, 131.1, 153.1, 153.4, 195.7. Anal.

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(C15H21NO4S) calcd %: C 57.85, H 6.80, N 4.50, S 10.30. Found %: C 57.50, H 6.91, N 4.42, S 10.58. [4-Methoxy-3-(pyridin-2-yldisulfanylmethyl)phenyl]carbamic Acid tert-Butyl Ester (7). Thioacetate (6) (102 mg, 0.33 mmol) was solvolyzed in a mixture of dry MeOH (4 mL) and NaOMe (80 mg, 4 equiv) for 4 h, at room temperature. The fraction of disulfide formed during the methanolysis was then reduced with TCEP (100 mg, 1 equiv) dissolved in a mixture of H2O (1 mL) and pyridine (600 µL). 2,2′-Dithiopyridine (480 mg, 6.6 equiv) was then added to this mixture and stirred overnight. After evaporation, the residue was dissolved in EtOAc and washed with water and brine and dried over MgSO4. The product was purified by silica gel chromatography (eluent: CH2Cl2 97/EtOAc 3), yielding 98 mg (79%) of a white powder, mp 122 °C. MS (ES-MS): M + H+ ) 379.2 [C18H22N2O3S2 (378.5)]. 1H NMR (CDCl3): δ (ppm) 1.50 (s, 9H), 3.81 (s, 3H), 4.01 (s, 2H), 6.26 (sbroad, 1H), 6.75 (d, J ) 8.8 Hz, 1H), 6.987.04 (m, 1H), 7.09 (d, J ) 2.7 Hz, 1H), 7.25 (m, 1H), 7.497.55 (m, 1H), 7.60 (dd, J ) 8.8 Hz, J ) 2.7 Hz, 1H), 8.40 (d, J ) 4.0 Hz, 1H). 13C NMR (CDCl3): δ (ppm) 28.3, 38.5, 55.7, 80.1, 110.9, 119.2, 119.7, 120.2, 122.0, 125.1, 131.1, 136.7, 149.1, 153.0, 153.3, 160.6. Anal. (C18H22N2O3S2) calcd %: C 57.11, H 5.86, N 7.40, S 16.94. Found %: C 57.01, H 5.92, N 7.40, S 16.91. UV: λmax ) 287 nm, ) 5900 M-1.cm-1. 4-Methoxy-3-(pyridin-2-yldisulfanylmethyl)benzenediazonium Trifluoroacetate (8). The aromatic amine of compound (7) (19 mg, 50 µmol) was deprotected in trifluoroacetic acid (2 mL) for 15 min. The mixture was cooled down to about 0 °C, and sodium nitrite (1.1 equiv) dissolved in water (100 µL) was added dropwise. The solution was evaporated to dryness, taken into H2O, and purified by reverse-phase HPLC on a Vydac C4 preparative column (22 × 250 mm; flow rate 15 mL.min-1; linear gradient: 0% B to 10% B in 5 min, then isocratic at 10% B. A: H2O, 0.1% TFA. B: CH3CN) yielding aryldiazonium (8) (26%). MS (ES-MS): M ) 262.87 (M - N2) [C13H12N3S2+ (290.4)]. 1H NMR (CD3OD): δ (ppm) 4.09 (s, 3H), 4.11 (s, 2H), 7.20-7.23 (m, 1H), 7.42 (d, J ) 9.3 Hz, 1H), 7.66-7.75 (m, 2H), 8.33-8.35 (m, 1H), 8.38 (d, J ) 2.6 Hz, 1H), 8.49 (dd, J ) 9.3 Hz, J ) 2.6 Hz, 1H). 13C NMR (CD3OD): δ (ppm) 37.1, 58.6, 103.0, 115.1, 121.8, 122.8, 131.2, 135.3, 137.5, 139.2, 150.3, 160.4, 160.9, 161.5, 169.3. [4-Methoxy-3-(pyridin-2-yldisulfanylmethyl)-5-[3H]-phenyl]carbamic Acid tert-Butyl Ester (7bis). Iodide (5) (44 mg, 0.1 mmol) was hydrogenated (P(3H2) ) 2 bar) for 1 day in EtOAc (1 mL) in the presence of Pd black (120 mg) and Et3N (60 µL). The mixture was centrifuged and the catalyst washed with EtOAc (5 mL). The organic phase was concentrated to 1 mL and purified by thin layer silica gel chromatography (eluent: n-hexane 8/EtOAc 2), yielding 18 mg (58%) of colorless oil, coeluting on TLC with the equivalent nonradioactive compound (6). The tritiated derivative was then dissolved in dry MeOH and reacted with an excess of NaOMe (15 mg) for 3 h, at room temperature. The mixture was then buffered by adding 75 µL of pyridine in 150 µL H2O. Treatment with an excess of TCEP (20 mg) reduced the formed disulfide. An excess of 2,2′-dithiopyridine (60 mg) was then added to this mixture, which was evaporated after 10 min. The remaining oil was diluted in 1.5 mL CH3CN and 750 µL H2O and purified by HPLC on a Vydac C18 analytical column (4.8 × 250 mm; flow rate 1 mL.min-1; isocratic: 55% B. A: H2O without TFA. B: CH3CN), yielding 61% of pure radioactive product (7bis). It coeluted with the non-radioactive compound on HPLC, and their 1H NMR spectra were identical. However, as expected, the integration of the proton in the ortho position of the methoxy group in 7bis corresponded to about 40% of the expected value on the non-radioactive compound, due to the replacement of part of the proton by tritium. Its radioactive specificity (12.5

Kessler et al.

Ci.mmol-1) was determined by UV spectroscopy (λmax ) 287 nm, ) 5900 M-1.cm-1) and β-counting and corresponded to the proton integration in 1H NMR. This compound was stable for months in a 1/4 mixture of CDCL3/toluene, in liquid N2. 4-Methoxy-3-(pyridin-2-yldisulfanylmethyl)-5-[3H]-benzenediazonium Trifluoroacetate (8bis). The aromatic amine of compound 7bis (3 µmol) was deprotected in trifluoroacetic acid (500 µL) for 2 h. The mixture was cooled down to about 0 °C, and sodium nitrite (1.1 equiv) dissolved in water (10 µL) was added. The solution was evaporated to dryness, taken into H2O, and purified by reverse-phase HPLC on a bondasorb C18 (10 µm) analytical column (4.8 × 250 mm; flow rate 1 mL.min-1; linear gradient: 0% B to 40% B in 20 min. A: H2O, 0.01% TFA. B: CH3CN) yielding aryldiazonium (8bis) (25%). The pure tritiated compound coeluted on a C18 column (conditions mentioned above) with the same non-radioactive derivative and had identical UV spectra. The concentrated HPLC fraction of compound 8bis was stable for months in liquid N2. UV: λmax ) 313 nm, ) 20 000 M-1.cm-1; the molar absorption coefficient was determined using the radioactive compound 8bis, by correlating its UV spectrum and its radioactive specificity (12.5 Ci.mmol-1). N-Terminal Modification of Naja nigricollis R33C Toxin Analogue with Desthiobiotin. After the automated peptide synthesis of the precursor of Naja nigricollis R33C toxin analogue (11), 6-fmoc-aminohexanoic acid was coupled to the N-terminus of the peptide still linked to the resin, using a standard HATU/diisopropylethylamine overnight activation in N-methylpyrrolidone. The fmoc group was released by piperidine treatment. Desthiobiotin was then coupled in the same way than the aminohexanoic linker. After deprotection for 1.5 h in TFA/triisopropylsilane/water (9/0.5/0.5), the peptide was precipitated in cold terbutyl ethyl ether and centrifuged. The pellet was dissolved in 10% acetic acid overnight. The linear peptide was purified as described earlier (11) and then refolded at 10 µM, in Tris-HCl 100 mM, pH 7.8, oxidized DTT 5 mM, for 1 day. Purification was carried out as described earlier (11) and afforded pure dtb-R33C toxin analogue, which was checked by electrospray mass spectrometry. Mcalcd ) 7043.2; Mfound ) 7043.0. Coupling of Aryldiazonium Salt (8bis) to dtb-R33C. A solution of 1.3 mg (190 nmol) of dtb-R33C in 250 µL of 1.6 M sodium citrate buffer (pH 2.2) was mixed, under rapid stirring, with 2.2 equiv of diazonium (8bis) diluted in 250 µL of the same buffer. The mixture was allowed to stand at room temperature in the dark for 1 day. The toxin derivative [3H]dtb-R33C-ArN2+ was then purified by HPLC on a Vydac C4 analytical column (4.8 × 250 mm; flow rate 1 mL.min-1; linear gradient: 10% B to 10% B in 10 min, then 10% to 18% B in 6 min, then 18% to 24% B in 19 min. A: H2O, 0.1% TFA. B: CH3CN), and checked by electrospray mass spectrometry. Mcalcd (dtb-R33C-ArN2+) ) 7222.25; Mfound ) 7222.35. UV: λmax ) 314 nm, ) 20 000 M-1.cm-1; toxin concentration was determined by acidic hydrolysis and subsequent quantification of the contained amino acids. Preparation of nAChR-Rich Membranes. nAChR-rich membranes from the electric tissue of Torpedo marmorata were prepared as previously described (13). The concentration of acetylcholine binding sites was typically between 0.9 and 1.4 nmol per milligram of proteins. Analytical Scale Photolabeling. Torpedo membranes (2 µM, 500 pmol R-bungarotoxin sites) were incubated in NaPO4 250 mM, pH 6.9, at room temperature in the dark for 15 min with [3H]-dtb-R33C-ArN2+ (0.95 equiv). The mixture was then stirred in a quartz cuvette and irradiated at 323 nm (200 µW.cm-2) for 10 min, at 10 °C. The size of the light beam was approximately 2 mm × 12 mm. For protection experiments,



Scheme 1. Synthesis of the Aryldiazonium Salts (8) and (8bis)a

a Reagents and conditions: (i) (a) BH3-THF, (b) Na2CO3, ICl, MeOH/H2O; (ii) (a) ClTrOMe, pyridine, 100 °C, (b) CH3I, NaHCO3, DMF; (iii) (a) NaSH, MeOH/THF, reflux, (b) tert-butyl dicarbonate; (iv) (a) THF, H2O, AcOH; (v) (a) CH3SO2Cl, Et3N, THF, (b) CH3COSH/Et3N, THF; (vi) H2 or 3H2 (2 bar), Pd black; (vii) (a) NaOMe, MeOH, (b) TCEP, (c) 2,2′-dithiodipyridine; (viii) TFA, NaNO2. Yields are given for non-radioactive products.

membranes were incubated with 20 equiv of R-bungarotoxin at room temperature for 1 h, before adding the tritiated photoactivatable toxin analogue. Following irradiation, the membranes were centrifuged at 22 000 g for 30 min, at 4 °C, and the pellets were solubilized in Laemmli buffer to be run on a 10% SDS-PAGE. The gels were then transferred to PVDFmembranes, which were exposed to a β-imager in order to visualize the labeled bands. These bands were compared to the one obtained by Western blotting of the same PVDF-membranes with mAbs 155, 165, and 140, which are specific for the R, γ, and δ subunits, respectively. Preparative Scale Photolabeling and Isolation of the Labeled r Subunit. Large quantities of torpedo membranes (30 nmol) were irradiated in the same conditions as for analytical scale experiments, except that the size of the light beam was 5 mm × 12 mm, and the energy was equal to 300 µW.cm-2. Following irradiation, the membrane suspension was centrifuged at 22 000 g for 30 min, at 25 °C, and the pellet solubilized in buffer A (Tris-HCl 50 mM, pH 8, LiDS 1%, LiCl 2 M). The mixture was then centrifuged as described before. The supernatant was poured onto streptavidin Sepharose beads (Amersham Biosciences), equilibrated with buffer A. The beads were washed with buffer A (2 × 10 mL) and with Tris 50 mM pH8, SDS 0.2% (3 × 10 mL). The labeled subunits were then recovered by incubating the beads twice (2 mL and 1 mL) in buffer B (Tris-HCl 2M pH 8, SDS 0.2%, biotin 50 mM) at 70 °C for 30 min. The subunits were reduced with TCEP (20 mM) at 25 °C overnight, and the sulfydryls were blocked with iodoacetamide (35 mM) at 25 °C for 15 min. The mixture was concentrated on centricon-30 and washed with Tris-HCl 20 mM pH 7. The solubilized and labeled subunits were subjected to 10% SDSPAGE. The gel was covered with a dry PVDF-membrane for 15 min, which enabled the location of the radioactive bands after exposing the membrane on a β-imager (Beta Imager 2000 from Biospace). The labeled subunits could then be excised from the gel without any staining. The gel fraction containing the R-subunit was cut into small pieces, transferred to an Eppendorf tube, and washed twice with a 50% aqueous solution of CH3CN (2 × 1 mL) for 10 min. “In Gel” CNBr Cleavage of the r Subunit and Peptide Purification. Cleavage of the R subunit and extraction of the labeled peptides were done as described in Grutter et al. (14), except that extraction of the peptides from the gel was carried out only with a mixture of H2O - 0.1% TFA/CH3CN (50/50). The extract was purified on a Vydac C4 narrow-bore column by HPLC (2.1 × 250 mm; flow rate 0.2 mL.min-1; linear gradient: 5% B to 5% B in 10 min, then 5% to 25% B in 10 min, then 25% to 35% B in 155 min, then 35% to 100% B in

30 min, then 100% to 100% B in 15 min, then 100% to 5% B in 5 min. A: H2O, 0.1% TFA. B: 70% CH3CN, 30% n-propanol, 0.095% TFA). UV absorbance was monitored at 220 nm. Fractions (400 µL) were collected, and aliquots were counted for radioactivity. Peptide Sequencing. HPLC fractions were concentrated (speedvac) and loaded onto a biobrene-pretreated filter of the sequencer. Automated Edman degradation was performed on an Applied Biosystems Procise sequencer. The HPLC output from each cycle was collected and counted for radioactivity quantification.

RESULTS Synthesis of the Bifunctional Photoprobe (8bis) (Scheme 1). The aryldiazonium salt (8) was synthesized in fourteen steps, with an overall yield of about 4%. A radioactive version was obtained by treating the aryliodide (5) with tritium gas leading to the thioacetate (6bis). Its radioactive specificity was 12.5 Ci.mmol-1. The main secondary reaction observed in this step (radioactive or not) was the concomitant desulfurization of (5). The methanolysis of the thioacetate (6bis) followed by the reduction of the formed disulfide and the dithiopyridinylation of the thiol were made one-pot. We thus obtained the precursor (7bis) of the bifunctional photoprobe (8bis). This precursor was kept in a mixture of toluene and chloroform (4/1) in liquid nitrogen and was stable for months. Diazotization of (7bis) led to the aryldiazonium salt (8bis), which was purified by HPLC. After concentration, the HPLC fractions were stable for months in liquid nitrogen. As was the case for a previous non-radioactive bifunctional photoprobe (12), this novel tritiated molecule carries an aryldiazonium salt, which can be photodecomposed at wavelengths which are safe for proteins (above 300 nm). Consistent with earlier data (15), replacing the dimethylamino group on the para position of the diazonium moiety (12) by a methoxy group (this work) shifted the maximal wavelength from 391 to 313 nm. Coupling of the Bifunctional Photoprobe to dtb-R33C. We synthesized a previously described analogue of the R-neurotoxin of Naja nigricollis (11) in which Arg33, at the tip of loop II, is replaced by a cysteine residue (Figure 1). This residue was shown to be a major hot spot for the interaction with the nAChR (16). Following automated peptide synthesis, we coupled manually a desthiobiotin through an aminohexanoic linker to the N-terminus of the peptide, in order to enable a prepurification of labeled subunits on streptavidin beads. In contrast to biotin, desthiobiotin binds reversibly to streptavidin (17), a huge advantage in such a study, as it enables the recovery of the purified labeled subunits.


Kessler et al.

Figure 2. UV/visible spectrum of compound (8) (......) and dtb-R33CArN2+ in water (;). Scheme 2. Coupling of Aryldiazonium Salt (8bis) to dtb-R33C Figure 1. Naja nigricollis R-neurotoxin. The structure has been taken from Zinn-Justin et al. (33). The molecular surface has been represented in yellow, showing the binding surface in blue and highlighting the critical Arg33 residue in red on tip of loop II. Note that the numbering of the residue corresponds to erabutoxin numbering.

We tried to couple the unique free cysteine of the refolded toxin to our bifunctional photoprobe at the same conditions as previously described for the achievement of R33C-DABD (12). Unfortunately, we obtained a mixture of the expected product and a molecule derived from the attack of the cysteine thiolate on the diazonium moiety (data not shown). This undesired reaction showed that, in the absence of light, the methoxy group renders the p-diazonium moiety more sensitive to nucleophilic attack than the dimethylamino group did. Lowering the pH from 7 to 4.5, during such coupling reaction, accelerates the disulfide exchange on the pyridyl disulfide, by protonation of the pyridine nitrogen (18). As the pKa value of 2,2′-pyridyl disulfide is 2.45 (18), we observed that decreasing the pH to 2.2 enabled the expected reaction to happen with the robust snake toxin, without any side reaction but with longer reaction times, as the cysteine residue is almost fully protonated. The maximum wavelengths of the obtained toxin photoprobe and of the initial bifunctional photoprobe (8 bis) were almost identical (Figure 2). This aryldiazonium toxin was stable for months in acidic conditions (HPLC fractions) in liquid nitrogen in the dark. However, the stability of the diazonium group at physiological pH (half-life ) 5 h at 17 °C in 250 mM sodium phosphate, pH 6.9, in the dark, data not shown) was not sufficient for equilibrium binding experiments in order to determine the inhibition constant of the modified toxin on the torpedo receptor. Nevertheless, a similar but more stable modified toxin (R33C-DABD), in which only the p-methoxy group on the bifunctional photoprobe was replaced by a p-dimethylamino moiety (12), showed an almost wild-type affinity for the nicotinic receptor. This novel toxin photoprobe had various specific characteristics (Scheme 2). The desthiobiotin tag enables purification of the labeled subunits of the nAChR. The disulfide bond, linking the toxin to the radioactive photoprobe, can easily be reduced, allowing the release of the toxin at will after photolabeling and, therefore, the transfer of radioactivity from the ligand to the receptor. This feature facilitates the purification and the analysis of the labeled peptides (10), as we have then to deal with standard labeling with a small non-peptidic molecule. The length

(8.5 Å) between the R-carbon attached to the photosensitive moiety and the reactive carbon on the arylcation, produced by UV irradiation, is almost as short as the replaced arginine residue, enabling a precise mapping of the binding site. It is also positively charged, therefore probably being a better mime than standard azido, diazirine, and benzophenone photoprobes on position 33 of the toxin. Photolabeling of Torpedo marmorata Membranes. The nAChR was irradiated in the presence of about 1 equiv of photosensitive toxin (dtb-R33C-ArN2+) at 323 nm, which is farther from the protein absorption band than the maximum wavelength of the photoprobe (314 nm). However, at this wavelength, the molar absorption coefficient is still close to the highest (Figure 2). For analytical experiments, labeled membranes were centrifuged, and the pellets taken in Laemmli buffer and run on a 10% SDS-PAGE. The gel was then transferred onto a PVDFmembrane. Once dry, the latter was exposed on a β-imager (Figure 3). Mainly four bands could be seen for the pellet dissolved in nonreducing Laemmli buffer (lane 5). Their apparent molecular weights were consistent with those of the


Figure 3. Detection of the nAChR labeled subunits by β-imaging. Torpedo membranes (2 µM R-bungarotoxin sites) were incubated with dtb-R33C-ArN2+ (0.95 equiv) and irradiated at 323 nm for 10 min, at 10 °C (see Experimental Procedures). Following irradiation, the membrane suspension was centrifuged at 22 000 g for 20 min at 4 °C, and the pellets solubilized in Laemmli sample buffer and run on 10% SDS-PAGE. Lane 1, molecular weight markers; lane 2, nonirradiated nAChR-photoprobe complex; lane 3, nAChR irradiated in the presence of dtb-R33C-ArN2+; lane 4, nAChR, preincubated with a large excess of R-bungarotoxin (20 equiv) and irradiated in the presence of dtbR33C-ArN2+; samples corresponding to lanes 1-4 were dissolved in reducing Laemmli buffer; lane 5, nAChR irradiated in the presence of dtb-R33C-ArN2+ (nonreducing Laemmli buffer). After SDS-PAGE, the proteins were transferred to a PVDF-membrane and exposed to a β-imager.

R, γ, and δ subunits coupled to a toxin molecule, with the highest weight being consistent with the dimer δ2 coupled to at least one toxin molecule. These two bands for the δ subunit show that, as is often the case, a mixture of monomers and dimers of nAChR were present in the torpedo membranes used. Three radioactive bands (lane 3) could be observed for the pellet dissolved in reducing Laemmli buffer. Their apparent molecular weights (38, 56, and 65 kDa) were consistent with those of the R, γ, and δ subunits, as expected after reduction of the disulfide bond linking the toxin to the receptor, leaving the radioactive probe on the receptor side. The labeling yield was determined on a similar polyacrylamide gel, which was cut in 1 mm pieces and counted for [3H]radioactivity. The band at 38 kDa was the most labeled (59% of the total labeling, 7.6% of the added toxin), due probably, in part, to the presence of two R subunits, followed by the band at 56 kDa (28% of the total labeling, 3.2% of the added toxin), with the last one being the band at 65 kDa, which was the least labeled subunit (13% of the total labeling, 1.2% of the added toxin). As expected, controls (i) with no irradiation of the toxinreceptor complex (lane 2) or (ii) with preincubation of the nAChR with an excess of R-bungarotoxin before the addition of dtb-R33C-ArN2+ (lane 4) showed no incorporation of radioactivity, demonstrating the total specificity of photolabeling. Desthiobiotin coupled to the N-terminus of the toxin analogue enabled purification on streptavidin beads of the whole set of labeled subunits (data not shown). Photolabeled membrane pellets were solubilized and denatured in lithium dodecyl sulfate, in order to purify the subunits covalently linked to the toxin by affinity chromatography on streptavidin Sepharose beads. Lithium chloride and lithium dodecyl sulfate were chosen instead of the sodium salts for solubility reasons, as sodium dodecyl sulfate precipitates rapidly in the presence of a high concentration of sodium chloride in buffers. Unexpectedly, recovery of the labeled subunits was not efficient enough when the disulfide bond linking the toxin to the irradiated photoprobe was directly reduced on the beads. To be quantitative, it required many repetitive reducing steps, preventing the recovery of the labeled subunits in a small volume. This was also the case when the beads were only incubated with an excess of biotin at room temperature. Thus, after many washings, the labeled subunits


Figure 4. Labeling specificity of dtb-R33C-ArN2+. After irradiation, the solubilized dtb-toxin-nAChR covalent complex was purified on streptavidin Sepharose beads and run on SDS-PAGE, using reducing Laemmli buffer. The proteins were then transferred from the gel to another PVDF-membrane, which was incubated with mAb155 + mAb165 or mAb155 + mAb140 to visualize the R and γ, respectively, the R and δ subunits of the Torpedo nAChR (see Experimental Procedures). Lanes 1 and 4, prestained molecular weight markers; lane 2, immunoblot with mAb155 + mAb165 (R + γ); lane 3, β-imaging of lane 2; lane 5, immunoblot with mAb155 + mAb140 (R + δ); lane 6, β-imaging of lane 5.

were recovered by heating the beads at 70 °C in the presence of an excess of biotin, and then reduced and alkylated. The radioactivity was hence transferred from the ligand to the subunits by release of the toxin from the photoprobe. The labeled subunits were then separated by 10% SDS-PAGE. On an analytical scale, the polyacrylamide gel was transferred on a PVDF-membrane, which was then exposed on a β-imager, followed by a Western blot with monoclonal antibodies mAb155, mAb165, and mAb140 directed against the R, γ, and δ subunits, respectively. This blot coupled to the [3H]-image demonstrated unambiguously that the labeled bands were indeed the formerly cited subunits (Figure 4). As expected, no labeling of the β subunit was observed. However, a varying amount of aggregates could be observed by β-imaging as well as by Western blotting. These aggregates depended on the quality of the torpedo membrane preparation but might also form in part during the treatment of the streptavidin beads at 70 °C. On a preparative scale (30 nmol of binding sites), the affinity purified subunits were run on three 1.5-mm-thick one-well SDSPAGE. The labeled subunits were detected without any staining. Actually, we covered the gel for about 15 min with a dry PVDFmembrane. This contact was sufficient to transfer enough molecules to be detected on a β-imager. Superimposing this image to the gel enabled cutting of the labeled subunits. Characterization of the Labeled Residues on the r Subunit. The gel fractions corresponding to the R subunit were digested “in-gel” with CNBr as described earlier (14). The extracted peptides were purified by HPLC on a Vydac C4 narrow-bore column (Figure 5). One major radioactive peak could be observed (fractions 39 to 44) corresponding to many poorly separated UV-detected peaks. The 4 fractions containing most of the radioactivity (40, 41, 42, and 43) were analyzed separately by sequential Edman degradation for 3H and mass release on 28 cycles (Figure 6). For fraction 40, 4 peptides beginning at RTrp118 (Io ) 124 pmol, R ) 86%), RGlu172 (first 6 residues detected, Io ) 73 pmol, R ) 58%), RLys179 (Io ) 81 pmol, R ) 88%), and RVal405 (Io ) 45 pmol, R ) 74%) could be detected. For fraction 41, five peptides from the nAChR R-subunit were detected: the first sequence began at RTrp118 (Io ) 50 pmol, R ) 92%), the second at RLys145 (Io ) 41 pmol, R ) 79%), the third at RGlu172 (first 20 residues detected, Io ) 39 pmol, R ) 83%), the fourth at RLys179 (Io ) 70 pmol, R ) 88%), and the fifth at RPro309 (Io ) 55 pmol, R ) 87%). For fraction 42, 4 peptides beginning at RGlu172 (first 25 residues detected,


Kessler et al.

harbor the critical C loop of the R-subunit, which occludes the ligand binding site and wraps agonists. However, only the peptide extending from RLys179 could be detected during the 28 cycles; the other peptide was already undetectable from cycles 7 and 11 for fractions 40 and 43, respectively. The 4 fractions also share 2 common positions of radioactive release, on cycles 12 and 20, corresponding to RTyr190 and RTyr198, respectively, on the peptide extending from RLys179. A third radioactive amino acid could be detected at cycle 14 for fractions 42 and 43, corresponding to RCys192 on the same peptide, as RAla343, which belongs to the peptide extending from RLys330, is cytoplasmic. As observed in fractions 40, 41, and 42, a very faint labeling could be seen on cycle 19 which might correspond to RTyr190 on the peptide beginning at RGlu172. Such an increase in radioactive detection was also visible on cycle 21, in fractions 42 and 43, and might correspond to RCys192 on the same peptide. It must be noted that a small radioactive peak was repeatedly detected at the third cycle. It would correspond to a faint labeling of RTyr181 on the peptide starting at RLys179 or of RGly174 on the peptide starting at RGlu172, which seems unreasonable considering their orientation and their distance to the ligand binding site (19). Moreover, repetitive experiments showed that the intensity of this peak was always low and not related to the intensity of the peaks at positions 12, 14, and 20 (data not shown). Further studies are necessary to explain its presence. Hence, the labeled residues are unambiguously primarily RTyr190, followed by RTyr198, with RCys192 being less labeled.

DISCUSSION

Figure 5. Reversed-phase HPLC of CNBr digests of the labeled R-subunit. Torpedo membranes (30 nmol, 2 µM R-bungarotoxin sites) were irradiated at 323 nm (300 µW.cm-2) for 10 min, at 10 °C in the presence of [3H]-dtb-R33C-ArN2+ (0.95 equiv). Following irradiation, the membrane suspension was centrifuged (22 000 g for 30 min, 25 °C), and the pellet solubilized in buffer A (Tris-HCl 50 mM, pH 8, LiDS 1%, LiCl 2 M). The labeled subunits were prepurified by affinity chromatography on streptavidin Sepharose beads (Amersham Biosciences) (6 mL), and the disulfide bonds were then reduced with TCEP and the cysteine blocked with iodoacetamide (see Experimental Procedures). The mixture was then concentrated on centricon-30 and subjected to 10% SDS-PAGE (three 1.5-mm-thick minigels). The labeled subunits were then excised from the gel, cut into small pieces, and cleaved “in gel” with CNBr. The extracted peptides were purified on a Vydac C4 narrow-bore column (2.1 × 250 mm; flow rate, 0.2 mL.min-1; linear gradient, 5% B to 5% B in 10 min, then 5% to 25% B in 10 min, then 25% to 35% B in 155 min, then 35% to 100% B in 30 min, then 100% to 100% B in 15 min, then 100% to 5% B in 5 min. A: H2O, 0.1% TFA. B: 70% CH3CN, 30% n-propanol, 0.095% TFA). UV absorbance was monitored at 220 nm. Fractions (400 µL) were collected, and aliquots were counted for radioactivity in a scintillation counter. (A) (-----) Gradient profile, (s) UV profile, (-b-) [3H] profile. (B) Zoom between fractions 36 and 47; (s) UV profile; 3 H profile has been represented as a histogram.

Io ) 28 pmol, R ) 89%), RLys179 (Io ) 63 pmol, R ) 87%), RPro309 (Io ) 116 pmol, R ) 85%), and RLys330 (Io ) 35 pmol, R ) 91%) were detected. For fraction 43, 3 peptides from the R-subunit were detected: the first primary sequence began at RGlu172 (first 10 residues detected, Io ) 24 pmol, R ) 93%), a second at RLys179 (Io ) 57 pmol, R ) 87%), and a third at RLys330 (Io ) 75 pmol, R ) 86%). As can be seen, the two unique peptides common to fractions 40, 41, 42, and 43 extend from RGlu172 and RLys179 and

Despite numerous photolabeling experiments of the muscletype nicotinic acetylcholine receptor with R-neurotoxins in these past decades (10, 20-29), no labeled residue belonging to the toxin-binding site could be characterized. The only success (10) managed a label-transfer technique with a commercial derivative. However, the linker between the photoactivatable moiety and the toxin was, at that time, too long to explore the binding site. Recently, we described the synthesis of a novel short cleavable photoactivatable probe with dual functionality, which can be selectively linked to any natural or engineered cysteinyl residue in peptides or proteins through a disulfide bond (12). This probe was coupled to a synthetic analogue of Naja nigricollis R-neurotoxin, in which a cysteine residue replaced Arg33 on the tip of loop II (Figure 1). Chemical synthesis of this toxin analogue allowed modification on a critical position, which could not be modified using wild-type toxins. This new photosensitive toxin analogue efficiently coupled to the receptor R subunit, but unfortunately, we could not get through the characterization of the labeled residues, as the synthesis of a radioactive version of this probe appeared impossible. We developed here a novel aryldiazonium photoprobe, in which a p-methoxy group replaced the stabilizing p-dimethylamino moiety (Scheme 1). This modification enabled the synthesis of a bifunctional cleavable tritiated photoactivatable probe via an iodinated derivative. The aryldiazonium salt showed long stability when stored in acidic conditions. The use of tritium makes it safe against irradiation and radioactively stable for years. However, the pH needed for coupling the aryldiazonium to the ligand restricts its use to acid-stable ligands. To our knowledge, no commercial molecule is shorter than 14 Å (compared to 8 Å here), and only one other short radioactive photocrosslinking/label-transfer probe was described (30). It harbors a benzophenone photosensitive moiety, is radioiodinatable, and has about the same length than our probe, but is more bulky. However, the authors did not get through the character-



Figure 6. Identification of nAChR R-subunit amino acids photolabeled by [3H]-dtb-R33C-ArN2+: 3H and mass release at each cycle upon sequential Edman degradation of HPLC fractions 40, 41, 42, and 43 (see Figure 5B). For fraction 40, 2.6 × 104 cpm was loaded on the filter. After 28 cycles, 16% of the radioactivity (4200 cpm) remained on the filter and 17% (4300 cpm) was recovered. Four peptides from the nAChR R-subunit were detected: the first primary sequence began at RTrp118 (Io ) 124 pmol, R ) 86%), a second at RGlu172 (Io ) 73 pmol, R ) 58%), a third at RLys179 (Io ) 81 pmol, R ) 88%), and a fourth at RVal405 (Io ) 45 pmol, R ) 74%). For fraction 41, 4.4 × 104 cpm was loaded on the filter. After 28 cycles, 17% of the radioactivity (7500 cpm) remained on the filter and 18% (7,800 cpm) was recovered. Five peptides from the nAChR R-subunit were detected: the first primary sequence began at RTrp118 (Io ) 50 pmol, R ) 92%), a second at RLys145 (Io ) 41 pmol, R ) 79%), a third at RGlu172 (Io ) 39 pmol, R ) 83%), a fourth at RLys179 (Io ) 70 pmol, R ) 88%), and a fifth at RPro309 (Io ) 55 pmol, R ) 87%). For fraction 42, 3.5 × 104 cpm was loaded on the filter. After 28 cycles, 14% of the radioactivity (4800 cpm) remained on the filter and 16% (5800 cpm) was recovered. Four peptides from the nAChR R-subunit were detected: the first primary sequence began at RGlu172 (Io ) 28 pmol, R ) 89%), a second at RLys179 (Io ) 63 pmol, R ) 87%), a third at RPro309 (Io ) 116 pmol, R ) 85%), and a fourth at RLys330 (Io ) 35 pmol, R ) 91%). For fraction 43, 2.4 × 104 cpm was loaded on the filter. After 28 cycles, 14% of the radioactivity (3300 cpm) remained on the filter and 15% (3600 cpm) was recovered. Three peptides from the nAChR R-subunit were detected: the first primary sequence began at RGlu172 (Io ) 24 pmol, R ) 93%), a second at RLys179 (Io ) 57 pmol, R ) 87%), and a third at RLys330 (Io ) 75 pmol, R ) 86%). The mass release curves were only represented for the two unique common peptides to fractions 40, 41, 42, and 43 extending from RGlu172 and RLys179. Their primary sequences are shown on top of the figures. Labeled residues are indicated in bold underlined characters.

ization of the labeled residues. Our probe showed also good labeling yields and is potentially dual, as the aryldiazonium moiety can be changed easily in an arylazido group, just by working in buffers containing sodium azide (12). The aryldiazonium photoprobe was subsequently coupled to a previously described (11) cysteine analogue of Naja nigricollis R-neurotoxin, through a cleavable disulfide bond. Moreover, to facilitate the purification of the labeled subunits, we tagged the N-terminus of the toxin with desthiobiotin. Assembly on the same toxin probe of a tag and a cleavable disulfide bond

linking the toxin to a radioactive photoprobe allowed us to characterize for the first time the labeled residues of the toxinbinding site on the R subunit of the nAChR. This tagged photoactivatable toxin labeled the torpedo nicotinic acetylcholine receptor with a 12% overall yield on the R (7.6%), γ (3.2%), and δ (1.2%) subunits, showing a small preference for the R subunit (3.8% on one R subunit at least). This may be due in part to the presence on this subunit of a higher number of aromatic amino acids, which are preferentially targeted by the arylcation formed through irradiation of an


aryldiazonium moiety. Edman degradation of purified CNBr fragments of the R subunit showed that three residues (RTyr190, RTyr198, and RCys192) were labeled. They all belong to the C loop, which wraps the agonists and occludes the binding site. They also belong to the group of residues labeled by another aryldiazonium antagonist named DDF (31, 32). But, in contrast to DDF, no labeling of RTrp149 could be detected, as we did not see any radioactive peak on cycle five when the CNBr fragments of HPLC fraction 41, containing a sequence beginning at RLys145, were submitted to Edman degradation. RTyr190, RCys192, and RTyr198 are located at less than 10 Å from the toxin backbone at position 33, as the reactive carbon of the photoprobe is located at about 8.5 Å, at most, from the Cys33 R-carbon. This is about the length of residue Arg33, meaning that the labeled residues are in close contact to Arg33 in the receptor/wild-type toxin complex. Our data corroborate double mutant cycle analysis with another short toxin (NmmI) (8), showing the proximity of RTyr190 and RTyr198 to Arg33. Notably, our results, obtained with a short R-neurotoxin, also fit well to the structure of the complex between a long neurotoxin and AChBP (4), where the shortest distances between the R-carbon of Arg33 and the side chains of the equivalent residues of RTyr190, RCys192, and RTyr198 on AChBP are close to 4-6 Å. This confirms a similar binding mode for short and long neurotoxins, at least at position 33, where the arginine residue may possibly act as a pivot. In contrast, our results do not agree with the recently proposed model of interaction between the short toxin NTII and the nAChR (9) in which the orientation of the toxin would hardly allow the labeling of RTyr190 and RCys192. In a prospective way, if we suppose that short- and longchain toxins bind in a similar manner, the AChBP/R-cobratoxin complex structure may also give a possible explanation of the different labeling yields between the γ and δ subunits, as (i) the common Trp55 would be too far away (>11 Å) to be labeled and (ii) γTyr116, which would be located as close to Arg33 as the three labeled R residues, is replaced by the nonaromatic δThr119, which is not a preferential target of aryldiazonium photoprobes. However, these conclusions need further work in the characterization of the γ- and δ-subunit labeled residues. The novel cleavable photoprobe described in this paper may be of great use for photocrosslinking/label transfer experiments between macromolecules and peptidic ligands. The literature shows no general rule to choose the best photosensitive moiety, when a photoaffinity labeling experiment on a given ligandreceptor complex is planned. Therefore, two and potentially three radioactive label transfer probes with various reactivity are welcome in the “photocrosslinking/label transfer kit”.

ACKNOWLEDGMENT We thank Dr. Tzartos for a generous gift of monoclonal antibodies directed against the nAChR subunits and Gilles Mourier for technical support and fruitful discussion on peptide synthesis.

NOTE ADDED AFTER ASAP PUBLICATION This manuscript was released ASAP on October 19, 2006 with missing information in the list of authors. The correct version was posted on October 25, 2006.

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