Agonist Binding Site of Torpedo Electric Tissue Nicotinic

THE JOURNAL OF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266. No. 33, Issue of November 25, pp. 22603-22612.1991 Printed in U.S.A.

Agonist Binding Site of Torpedo Electric Tissue Nicotinic Acetylcholine Receptor A NEGATIVELY CHARGED REGION OF THE 6 SUBUNIT WITHIN 0.9 nm OF THE a SUBUNIT BINDING SITE DISULFIDE*

(Received for publication, July 11, 1991)

Cynthia CzajkowskiSslI and Arthur KarlinS$II-’;* From the $Center for Molecular Recognition and the Departments o/ §Biochemistry, 11 Physiology, and )I Neurology, College of Physicians and Surgeons, Columbia University, N e w York, New York 10032

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by a C ~ S ’ ’ ~ and Cyslg3 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and a Cys’” or CyslS3. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked 13H]N- glycylcysteamine could react with carboxyls within 0.9 nm of C y ~ ’ @ ~ / C y s ’ ~ ~ . To promote amide bond formation between the tethered amino group and receptor carboxyls, we added l-ethyl-3-(3’-dimethylaminopro- py1)-carbodiimide. The predominant sites of amide coupling were on the 6 subunit, in CNBr fragment 4 (6 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Glu residues in this region. One or more of these residues is likely to contribute to the binding of ACh.

~ ~ ~~~~

The first step in the function of nicotinic ACh’ receptors is

*This research was supported in part by Research Grant (NS- 07065) from the National Institutes of Health and from the Muscular Dystrophy Association, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Partially supported by a National Institutes of Health Training Grant NS-07258.

** To whom correspondence should be addressed Center for Mo- lecular Recognition, Columbia Univ., 630 w. 168th St., New York, NY 10032. Tel.: 212-305-5778.

The abbreviations used are: ACh, acetylcholine; ACP, S-(2-acetamidoethyl)dithio-2-pyridine; BAC, bromoacetylcholine bromide; Boc-Gly-OSu, t-butyloxycarbonyl-glycine-N-hydroxysuccinimidyl ester; BuAc, upper phase of n-butanol-water-acetic acid (4:5:1); ChMeAc, chloroform-methanol-acetic acid (45:4:1); CP, S-(2-amino- ethyl)dithio-2-pyridine; DTT, dithiothreitol; [3H]GC, [3H]N-glycylcysteamine; [‘HIGCP, S-(2-[3H]glycylamidoethyl)dithio-2-pyridine; HFBA, heptafluorobutyric acid; EPCD, l-ethyl-3-(3’-dimethylami- nopropyl) carbodiimide; MBTA, N-(4-maleimido)benzyltrimethylammonium iodide; NEM, N-ethylmaleimide; PySSPy, 2,2’-

the binding of ACh. A characteristic property of the ACh binding site is a readily reducible disulfide bond about 1 nm from a negative subsite (Karlin, 1969). The latter was postu- lated to bind the positively charged quaternary ammonium group of ACh and other potent agonists and competitive antagonists. The binding site disulfide was shown by affinity labeling and peptide mapping to be formed between adjacent cysteinyl residues, C ~ S ’ ’ ~ and CysIg3, in the a subunit from Torpedo californica ACh receptor (Kao et al., 1984; Kao and Karlin, 1986). A homologous pair of adjacent Cys residues is found in all sequenced a subunits, both muscle-type and neuronal-type ACh receptors (reviewed by Claudio, 1989; Boulter et al., 1990; Schoepfer et al., 1990; Karlin, 1991; and Galzi et al., 1991).

Torpedo a subunit residues Tyr’” (Dennis et al., 1988; Abramson et al., 19891, Trp14’ (Dennis et al., 1988), and Tyrg3 (Galzi et al., 1990) were also located at the ACh binding site by affinity labeling and peptide mapping. No aspartyl or glutamyl residue, however, has yet been located at the ACh binding site. It has been suggested that ionized Tyr phenolic side chains (Galzi et al., 1990) or the electron-rich aromatic rings of Tyr, Trp, and Phe side chains (Dougherty and Stauf- fer, 1990) may contribute to the binding of the ACh quaternary ammonium group and hence could form the negative subsite.

The electrophilic or photoactivated affinity labels used in the work cited above might not react with a carboxylate even if one were present at the ACh binding site. By contrast, a reaction which would result in a stable bond with a carboxyl group is the carbodiimide-induced formation of an amide bond between a carboxyl and a primary amine (reviewed by Means and Feeney, 1971). We therefore synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine ([3H]GCP); i.e. N-[3H]glycylcysteamine ([3H]GC) in a disulfide with 2-mercaptopyridine (see Scheme 1). The latter is an excellent leaving group, promoting disulfide formation (Brocklehurst and Little, 1973; Chong and Hodges, 1981) between [3H]GC and protein sulfhydryls. In the extended conformation of [3H]GC, the distance between the cysteamine thio group and the glycyl amino group is 0.9 nm. Using [3H]GCP and l-ethy1-3-(3’-dimethylamino- propy1)carbodiimide (EPCD), we have cross-linked a CysIg2/ CysIg3 to carboxyls on the 6 subunit. One CNBr fragment of 6 was the predominant site of amide cross-linking of [3H]GC and thus contains Asp and/or Glu residues likely to contribute to the negative subsite of the ACh binding site.

dipyridyl disulfide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid TLC, thin-layer chromatography; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid.

22603

22604 6 Subunit Carboxyl Close to ACh Receptor Binding Site

EXPERIMENTAL PROCEDURES

Materials The following chemicals and radiochemicals were obtained com-

mercially as follows: 2,2'-dipyridyl disulfide, 2-mercaptopyridine, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N-hydroxysuc- cimide, di-f-butyl dicarbonate, and acetic anhydride from Aldrich Chemical Co.; cysteamine, cystamine, l-ethyl-3-(3'-dimethylamino- propy1)carbodiimide (EPCD), l-cyclohexyl-3-(2-morpholinoethyl)- carbodiimide (HMCD) from Sigma Chemical Co.; trimethylamine in methanol from Eastman Kodak Co., deuterated methanol from Merck Chemical Co., ["Hlacetic anhydride from Amersham Corp., and [Z- ,"H]glycine and [cu-12sII]bungarotoxin from NEN Research Products- Dupont Co. Reagent grade and HPLC grade solvents were used as received. Fatty acid-free bovine serum albumin was from Sigma Chemical Co., NCS solubilizer was from Amersham Corp., PPO and dimethylPOPOP were from Packard Instruments. Affigel 401 was from Biorad Corp. Aluminum sheetbacked silica gel, 60 F,,,, from EM Science, Cherry Hill, NJ, was used for analytical TLC, and the same silica gel (0.5 to 3 mm thick) on glass plates was used for preparative TLC. TLC was developed with the upper phase of n- butanol/water/aceticacid(4:5:1) (BuAc)orwithchloroform/methanol/ acetic acid (45:4:1) (ChMeAc). Bromoacetylcholine bromide (BAG) was synthesized following Damle et al. (1978) and N-(4-maleimido)benzyltrimethylammonium iodide (MBTA) was synthesized as in Karlin (1977).

Synthesis of 2-Mercaptopyridine Derivatives S-(2-AminoethylJdithio-2-pyridine (CP)-7.5 ml of 0.5 mM 2,2'-

dipyridyl disulfide (PySSPy) in n-propanol, 0.75 ml of 1 M cysteamine-HC1 in methanol, and 0.125 ml of 4.2 M trimethylamine in methanol were mixed and kept at room temperature for 45 min. The mixture was protected from direct light. We added 25 ml of 0.2 M sodium acetate/acetic acid (pH 5.0) and extracted the mixture with diethyl ether (20 X 40 ml). The aqueous phase was cooled in ice, brought to pH 9 with 3 ml of 0.67 M sodium carbonate, and extracted with dichloromethane (4 X 30 ml). The extract was kept on ice, dried with anhydrous magnesium sulfate, rotary-evaporated under argon, recovered in about 3 ml of dichloromethane, and stored under argon at -20 "C. We analyzed this crude mixture by TLC on silica gel, developed with BuAc. The pyridyl group was detected with short UV, and cysteamine was detected with ninhydrin spray. The mixture consisted mostly of a new product of Ri = 0.4, containing the pyridyl disulfide group and a free amino group, which we tentatively identified as CP. Also present were small amounts of PySSPy, 2-thiopyridone, and cystamine, which we identified by comparison with the authentic compounds. We estimated the concentration of CP as the concentration of 2-thiopyridone, determined by absorbance before and after reduction by 20 mM dithiothreitol in 100 mM sodium borate, pH 8.0. The molar extinction coefficient of PySSPy at 281 nm is 9700 cm" M", and that of 2-thiopyridone a t 343 nm is 7060 Cm"M" (Brockle- hurst and Little, 1973). We recovered 0.29 mmol of CP.

S-(2-Acetamidoethylldithio-2-pyridine-To 7.4 pmol of crude CP in 200 pl of ethyl acetate, we added 2 p1 of neat acetic anhydride (21 pmol). The mixture was kept a t room temperature for 1 h. It was dried, dissolved in 100 p1 of dichloromethane, dried, and dissolved in 200 of acetonitrile. By TLC, as above, the product consisted mostly of a ninhydrin-negative, UV-absorbing component with an R/ = 0.7. There was a small amount of PySSPy present. One-half of the product was applied to a 2-mm-thick silica gel plate, and developed with BuAc. A UV absorbing band a t R, = 0.7 was cut out, extracted with ethyl acetate, filtered, dried, and dissolved in ethyl acetate. By absorbance, 1.3 pmol was recovered. This was repeated on a 5-fold larger scale. ACP (0.5 mg) was dissolved in deuterated methanol. Its 'H NMR spectrum (Bruker 500 MHz) was consistent with the proposed structure. Mass spectrometry (Hewlett Packard 5988A quadrapole MS with 5890 GC) of ACP after chemical ionization yielded (M + 1)+ of mass 229, also consistent with its proposed structure.

S-(2-~H]Acetamidoethyl~dithio-2-pyridine-['H]Acetic anhydride (3.35 pmol, 25 mCi) was transferred in 100 p1 of dichloromethane from a break-seal tube to a reacti-vial (Pierce) containing 15 pmol of C P in 240 pl of dichloromethane. The vial was sealed and kept at room temperature for 1 h. Nonradioactive acetic anhydride (30 pl of 1 M acetic anhydride in dichloromethane) was added. After reaction for 2 h, the reaction mixture was dried under a gentle stream of nitrogen. The residue was dissolved in 100 pl of dichloromethane, dried, dissolved in 200 pl of dichloromethane, and stored a t -70 "C.

The product was purified by preparative TLC on a 4- by 10-cm plate of 3-mm-thick silica gel developed with BuAc. A band with an R/ of about 0.7 was cut out and extracted with ethyl acetate (4 X 2 ml). The extract was stored a t -70 "C. A small amount of precipitate that formed was pelleted and discarded. By absorbance, we recovered 10 pmol of [3H]ACP with a specific activity of about 380 Ci/mol. This product gave a single spot with R, = 0.7 on analytical TLC, as did the nonradioactive ACP.

S-~2-~H~Glycylamidoethyl)dithio-2-pyridine-[2-3H]Glycine(lO5.3 nmol, 5 mCi) in 0.01 N HCI was transferred to a reacti-vial and dried in a Savant Speed-Vac. Nonradioactive glycine (8.43 pmol) in 40 pl of 0.5 N NaOH was added with stirring at 4 "C. To protect the amino group, we added 11 pmol of di-tert-butyl dicarbonate in 40 pl of dioxane and stirred the mixture overnight a t room temperature. We added 100 pl water and extracted the mixture twice with ethyl acetate (2 X 200 pl). The aqueous phase was collected, cooled on ice, and brought to pH 2-3 with 5 p1 of 6 N HCl. The mixture was extracted once with 200 pl and twice with 100 pl of ethyl acetate. The organic phase was collected and washed with ice-cold 5% HCl (3 X 100 pl) and with ice-cold saturated NaCl (2 X 100 pl). The ethyl acetate phase was dried with anhydrous sodium sulfate, and the mixture was centrifuged. The supernatant was collected, and the pellet was rinsed with ethyl acetate (2 X 100 pl). The ethyl acetate (supernatant plus rinses, -400 pl) was dried in Speed-Vac, and the residue was dissolved in 50 pl of anhydrous dimethoxyethane.

To activate the carboxyl group, we added 20 pmol of N-hydroxysuccinimide, 20 pmol of dicyclohexylcarbodiimide, and 5 pmol of 4- dimethylaminopyridine in 61 p1 of dimethoxyethane. The mixture was stirred overnight at room temperature. A white precipitate was pelleted. The pellet was rinsed with dimethoxyethane (2 X 50 pl), and the washes added to the first supernatant, giving a total volume of about 200 p1. We analyzed the reaction mixture by TLC developed with ChMeAc. The reaction mixture consisted mostly of t-butyloxy- carhonyl[2-3H]gly~ine-N-hydr~~y~~~cinimidyl ester (B~c- [~HlGly - OSu) (R, = 0.70), identified by comparison with authentic Boc-Gly- OSu (Bachem Bioscience Inc.). Also present in the reaction mixture were N-hydroxysuccinimide (R/ = 0.29) and 4-dimethylaminopyridine

T o synthesize ['HIGCP, we added the crude B O C - [ ~ H ] G ~ ~ - O S U (200 p1) to 15 pmol of dried CP. Triethylamine (1 p l ) was added, and the mixture was stirred overnight a t room temperature. The products were analyzed by TLC, as above. A new product with an R, = 0.57 was identified as Boc-[~H]GCP by the following criteria. 1) The product was ninhydrin-negative unless first incubated in TFA vapors to remove the t-butyloxycarbonyl group. 2) The spot stained positive with an iodine-azide reagent specific for thiols and disulfides (Smith, 1969). 3) The compound was disulfide-coupled to 2-thiopyridine as determined by absorbance after reduction by 20 mM dithiothreitol. 4) The compound was radioactive and thus contained [2-3H]glycine. Also present on the plate were small amounts of PySSPy, N-hydroxysuccinimide, CP, and 4-dimethylaminopyridine. We purified Boc- ['HHJGCP by TLC on a 0.5-mm thick silica gel plate, developed with ChMeAc. The product was detected by short UV, scraped off the plate, extracted with methanol, dried, and dissolved in 100 pl of dichloromethane. T o deprotect Boc-[~H]GCP, we added 100 pl of 100% TFA and incubated the mixture for 50 min at room temperature. The sample was dried and dissolved in 200 p1 of methanol. We estimated the concentration of [3H]GCP by absorbance. We recovered 1-3 pmol of ['HIGCP with a specific activity of 0.3-1.0 Ci/mmol.

(R/ = 0.06).

Miscellaneous Methods Protein was determined by the method of Lowry et al. (1951) with

fatty acid-free bovine serum albumin as a standard. Toxin binding was assayed with 12sII-cu-bungarotoxin according to Czajkowski et al. (1989). ACh receptor in Triton X-100 solution was purified by affinity chromatography on the adduct of bromoacetylcholine and Affi-Gel 401 as previously described (Reynolds and Karlin, 1978). Approxi- mately 80% of the toxin binding sites were associated with the disulfide-cross-linked dimeric form. SDS-polyacrylamide gels consisted of a 4% stacking gel over a 7.5% resolving gel (Laemmli, 1979) unless stated otherwise. Following electrophoresis, the gel was stained with 0.01% Coomassie Brilliant Blue in 25% isopropanol/lO% acetic acid and destained in 7% acetic acid/lO% methanol. For gel slicing, individual lanes were cut into 1-mm slices which were treated with NCS solubilizer and counted in 2,5-diphenyloxazole/1,4-bis[2-(4- methyl-5-phenyloxazolyl)]benzene/toluene scintillant. For electroelution, the stained band corresponding to the desired subunit was cut

6 Subunit Carboxyl Close to ACh Receptor Binding Site 22605

out with a razor blade, washed in 50 mM ammonium bicarbonate/ 0.1% SDS (electroelution buffer), transferred to an electroelution cup (ISCO), and electroeluted overnight a t 50 V. Automated Edman degradation was performed on a Applied Biosystem 477A sequencer.

RESULTS

Reaction of pH]GCP with Mildly Reduced Receptor-A disulfide bond between a C Y S ' ~ ~ and Cyslg3 at the ACh binding site is unusually susceptible to reduction (Kao and Karlin, 1986), and we used this property to direct [3H]GCP to these Cys residues. Before reducing this disulfide, we alkylated all accessible sulfhydryls with 20 mM NEM; this was sufficient to prevent any reaction of [3H]GCP with unreduced ACh receptor. We then reduced the receptor with 0.2 mM DTT at pH 8.1 for 20 min. These mild conditions completely reduced a Cy~'~~/Cys '~ : ' (Kao and Karlin, 1986) and partially reduced the disulfide bond between 6 subunits in receptor dimer (Ham- ilton et al., 1979). No other receptor disulfides were signifi- cantly reduced under these conditions (Kao and Karlin, 1986). ["HIGCP reacted with the mildly reduced receptor predominantly on the a subunits (Fig. U): 82 f 3% of the tritium recovered in the subunits was in a, 3.7 f 0.5% was in 0, 4.6 f 0.4% was in y, and 9.8 f 2.0% was in 6 ( n = 4). The mean extent of incorporation was 5.6 f 0.8 nmol/mg receptor ( n = 4); hence, about 4.6 nmol of ['HIGC per mg receptor was incorporated into the a subunits.

The presence of an agonist, either acetylcholine, carbamylcholine, or phenyltrimethylammonium, during the mild reduction and subsequent reaction with [3H]GCP inhibited the incorporation of ['HH]GC into a (Table I). Furthermore, reacting either MBTA or BAC, two highly specific affinity labels of the reduced ACh binding site, with the reduced ACh receptor inhibited the subsequent reaction of ['HIGCP with a (Table I).

The [3H]GC incorporated into mildly reduced receptor was completely removed by 50 mM DTT, consistent with the attachment of [3H]GC via a disulfide bond. Also, S-(2-[3H] acetamidoethyl)dithio-2-pyridine ( [3H]ACP), a compound similar to ['HIGCP, but lacking a primary amino group, reacted with the reduced receptor just as did [3H]GCP (data not shown).

a-Subunit, disulfide-linked to [3H]GC, was cleaved with CNBr, and the fragments were separated by HPLC (Kao et al., 1984). There was a predominant peak of radioactivity (Fig. 2 4 ) . Automated Edman degradation of this peak yielded the N-terminal sequences of CNBr 6 (a 179-207) and CNBr 4 ( a 145-171) (Table 11). The major component, CNBr 6, contains Cys'g2 and Cys"" and no other Cys residues. CNBr 4 contains no Cys residues.

Most of the tritium associated with CNBr 6 was released in the first cycle of Edman degradation (Fig. 2B). The origin was ['HIGly, cleaved from the N-terminus of [3H]gly~y1~y~- teamine disulfide-linked to CNBr 6. Correspondingly, Gly was detected in the first cycle of Edman degradation (Table 11, columns 6 and 7). Despite the loss of most of the attached ["HIGly, there was a small but significant rise in the tritium released in cycles 14 and 15, corresponding to C Y S ' ~ ~ and Cyslg3 (Fig. 2B).

Amide Coupling of Disulfide-linked fH]GC Induced by EPCD-['H]GC tethered by a disulfide to a has a primary amino group at the other end of the molecule. We used carbodiimides to couple this amino group with neighboring carboxyl groups (Scheme 1). We assayed for this amide coupling of ['HIGC by determining the fraction of [3H]GC initially attached by a disulfide bond that remained attached after exhaustive reduction by DTT in SDS. EPCD, l-cyclohexyl-3,2-morpholinoethyl carbodiimide, and dicyclohexyl-

0 40 80 120 180

aooo 1 0 40 a0 120 180 0 40 00 120 180

SLICE NUMBER

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of receptor subunits linked to [3H]GC by a disulfide bond before EPCD and by an amide bond after EPCD. In A and B, the receptor was reduced with a low concentration of DTT and then reacted with [3H]GCP. In B the receptor was further reacted with EPCD and then exhaustively reduced with DTT in SDS. In C and D, the receptor was first reduced with a low concentration of DTT, the reduced cysteines (mainly on a) were blocked with N - ethylmaleimide, and the receptor was reduced with a higher concentration of DTT, completely reducing the 6-6 disulfide, and then reacted with [3H]GCP. In D, the receptor was further reacted with EPCD and then reduced with DTT in SDS. In detail, purified ACh receptor (8 nmol of ACh binding sites a t 2.7 FM) in TNPlOO (0.2% Triton X-100, 100 mM NaCl, 10 mM NaPO,, 1 mM EDTA, 3 mM NaNB) was reacted for 30 min at room temperature with 200 p~ diisopropylfluorophosphate, to inhibit proteolysis, and with 0.1 pCi of [l4C]NEM, to provide a marker for recovery. Subsequently, the receptor was reacted with 20 mM NEM for 2 h, to block all accessible sulfhydryls, and then dialyzed against 0.2% Triton X-100, 1 mM EDTA, 50 mM Tris, pH 8.1, a t 4 "C for 4 h, to change the pH and to remove excess reactants. This receptor was used in the following reactions. A, 1.5 nmol of receptor in approximately 50 pl was kept for 1 h at room temperature, reacted with 0.2 mM DTT for 20 min, dialyzed against TNP50 (same ingredients as TNP100, except 50 mM NaC1) for 4.5 h a t 4 "C, reacted with 15 nmol of ['HH]GCP for 1 h at 25 "C, reacted with 5 mM NEM for 15 min, and dialyzed against 0.2% Triton X-100, 10 mM MOPS, pH 6.3, 4 "C for 4.5 h. (This is where B diverges from A . ) This receptor was kept for 3 h at 25 "C, treated with 20 mM glycine methyl ester for 10 min, precipitated with 90% acetone, dried, dissolved in 2% SDS, 20 mM Tris, pH 8.1, kept for 30 min at 50 "C, acetone-precipitated again, dissolved in Laemmli sample buffer (no reducing agent), and applied to a 1.5-mm polyacrylamide slab gel with a 7.5% resolving gel. After electrophoresis, staining, and destaining the individual lanes were cut into 1-mm slices and counted. B, receptor was reduced and labeled with [3H]GCP as in A, but after the dialysis against Triton X-100, MOPS buffer, it was reacted with 100 p~ EPCD for 3 h a t 25 "C. It was treated with 20 mM glycine methyl ester, precipitated with 90% acetone, and dried as in A. It was dissolved in 2% SDS, 20 mM Tris, pH 8.1, containing 50 mM DTT, kept at 30 min at 50 "C to reduce all disulfides, reacted with 110 mM NEM, and then acetone-precipitated, dissolved in Laemmli sample buffer, and electrophoresed, sliced, and counted in parallel with A. C, 1.5 nmol of receptor was reduced with 0.2 mM DTT for 30 min at room temperature and reacted with 1 mM NEM for 30 min to reduce and alkylate the binding site cysteines and reacted with 2 mM DTT for 20 min. I t was then dialyzed, labeled with (3H]GCP, and analyzed as in A . D, the receptor was mildly reduced, blocked with NEM, further reduced, and reacted with ['HI GCP, as in C. It was then reacted with EPCD, exhaustively reduced, and analyzed as in B. Tritium counts loaded on the gels and recovered in the slices were: A, 121,000, 112,000; B, 18,000, 15,000; C, 50,000, 53,000; D, 7,800, 7,200.


TABLE I Block of the reaction of PHjGCP with the a subunit

Blocking agent Incorporation as fraction of con-

trol

% 8 f 1

1 9 f 2 50

~~

Acetylcholine, 0.2-1 mMu Carbamylcholine, 20 mMa Phenyltrimethylammonium, 10 mMa Bromoacetylcholine, 10-20 p ~ * 44 f 10 Maleimidobenzyltrimethylammonium, 8 ? 0.5

10-20 pMh

"Present during both the reduction with dithiothreitol and the

I, Added after the reduction with dithiothreitol. reaction with ["HJGCP.

h

10000 ' I ' 1 " ' " '

c! .- R A E BOO0

6, 6000

4000 / I

0 20 40 60 BO 100

ELUTION TIME (min )

i O I , , . , . , , , 1

0 4 6 12 16 20

CYCLE

FIG. 2. A, HPLC of [3H]GC disulfide-linked to the a subunit. Receptor was mildly reduced, reacted with [3H]GCP, and electrophoresed as in Fig. 1A except that the sample was loaded for electrophoresis in one large well. The band corresponding to the a subunit was cut out, electroeluted, and cleaved in 100 p1 of 1 M CNBr in 80% TFA for 12 h a t room temperature (DiPaolo et ai., 1989). 25 p1 of the cleavage mixture was injected directly onto a Vydac C18, 25- X 0.46- cm column. Peptide fragments were eluted with a mixture of 0.2% HFBA (buffer A) and 60% acetonitrile, 40% isopropanol, 0.2% HFBA (buffer B) in a linear gradient of 5-80% buffer B over 100 min (Kao e t ai., 1984). The flow rate was 1 ml/min, and fractions were collected every minute. The tritium in 50-pl aliquots is shown. Input of tritium was approximately 716,536 cpm; 692,358 cpm was recovered. B, fraction 55 from the above HPLC was subjected to automated Edman degradation with the following modification. The ATZ amino acid conversion to phenylthiohydantoin derivative was omitted and the freshly cleaved ATZ amino acids were directly transferred to the integral fraction collector. For each sequenator cycle, the radioactivity of the ATZ amino acids recovered was measured by evaporating the residual butyl chloride remaining in the fraction collector tubes to dryness, extracting the contents of each tube two times with 100 pl of methanol, and counting the methanol. The individual tubes were also counted to ensure that the methanol extraction was efficient. The Polybrene-coated disc and disc support were counted to determine radioactivity remaining at the end of the sequencing run. 100,700 cpm were applied. 1% of applied radioactivity was still present on the disc and disc support a t termination of sequencing run. 22,012 cpm were recovered in the fractions.

carbodiimide were all effective in inducing this amide coupling of ["HIGC. We used EPCD routinely. After 3 h of reaction with 1 mM EPCD at pH 6.1 and 30 "C, 45% (n = 3) of initially disulfide-linked ['HIGC was amide-coupled to the receptor.

TABLE I1 N-terminal sequences of CNBr fragments of a in the major HPLC

peak of disulfide-linked PHjGC Fraction 54 from the HPLC represented in Fig. 2 was subjected to

automated Edman degradation. The residues released corresponded to two CNBr fragments of a; CNBr 6, a 179-207, and CNBr 4, a 145-171. The first 20 residues of these two fragments are listed in columns 2 and 4, followed in each case by the quantity released or "- " if not detected. Column 6 headed "Other" contains the residues released not corresponding to a predicted a fragment.

CNBr 6 CNBr 4 Other Cycle

AA Quantity AA Quantity AA Quantitv

1 K 21 K 2 D 30 L 3 Y 45 G 4 R I 5 G 33 W 6 W T 7 K 22 Y 8 H 58 D 9 w 8 G

10 v 8 T 11 Y 14 K 12 Y 15 V 13 T 39 S 14 C I 15 C S 16 P 7 P 17 D 10 E 18 T 15 S 19 P 8 D 20 Y 2 R

Pmol 2 1 5

20 10

6 14 5

16

7 5 9 7 2

G 19 S L

11 9

E 12 A 5 R 13 T 29

S- 7

I 3 E 4

W 8

p $ H ; coo-

Lr' LT-l &PC. OCNH~S-M co 0:::; e p;&

SCHEME 1. The structure of S-(2-['H]glycylamidoethyl)dithio-2- pyridine and its projected reactions at the ACh binding site. PS is the 2-mercaptopyridine leaving group and -SRNH, is the ['Hlgly- cylcysteamine moiety. The asterisk represents the 'H label. Where two reagents are added, NEM is added second.

Under the same conditions, none of N-[3H]acetylcysteamine initially disulfide-linked to the receptor was amide-coupled by EPCD; i.e. the free amino group of glycylcysteamine was necessary for EPCD-induced coupling.

Although 1 mM EPCD effectively amide-coupled ['HIGC to the receptor, it (and the other carbodiimides as well) also cross-linked the receptor subunits directly; i.e. independently of [3H]GC. No monomeric subunits were seen after SDS- PAGE. The receptor subunits were cross-linked by nonreduc- ible bonds to form high molecular weight complexes, and this cross-linking was independent of the presence of [3H]GCP. More useful results were obtained under milder reaction conditions: after 3 h of 100 p~ EPCD, at pH 6.3 and 25 "C, 18 k

6 Subuni t Carboxyl Close to ACh Receptor Binding Site 22607

FIG. 3. Receptor subunits separated by SDS-PAGE identified by immunoblotting. Receptor was subjected to the same series of reactions and treatments as in Fig. lB, and identical samples of treated receptor were subjected to SDS-PAGE. Four replicate lanes were electroblotted onto nitrocellulose paper and probed with antibodies following the methods of Towbin et al. (1979). The blots were soaked in 5% bovine serum albumin, 0.9% NaCI, 10 mM Tris, pH 7.4 (bovine serum albumin-NaC1-Tris), and then incubated for 2 h a t room temperature with subunit-specific monoclonal antibodies (dialyzed, ammonium sulfate cuts of concentrated hybridoma culture medium). The blots were washed with four changes of bovine serum albumin-NaC1-Tris and reacted for 1 h with goat-anti-rat IgG con- jugated to horseradish peroxidase. After further washing, 0.04% 4- chloro-1-naphthol and 0.01% H202 were added to the blots. After 15- 30 min, the blots were washed, dried, and photographed. In lane I , 2.5 nm anti-n mAb142 was used; in lane 2, 5.2 nM anti+' mAbll l ; in lane 3, 6.7 nM anti-y mAb168; and in lane 4, 1.2 nM an t i4 mAb166. All monoclonal antibodies were given to us by Dr. Jon Lindstrom and are described in Tzartos et al. (1986). The locations of unmodified receptor subunits, run in parallel and stained with Coomassie Bril- liant Blue, are indicated.

400 A

300

400 E

B ACh t

300 JI t

0 30 60 80 120 150

SLICE NUMBER FIG. 4. Effect of ACh binding-site ligands on the EPCD-

induced amide coupling of ["HIGC. Receptor was treated as in Fig. lR, except that following dialysis against Triton-X-lOO/MOPS buffer, the receptor was incubated either for 45 min in buffer (A ), or in 500 PM diisopropylfluorophosphate for 15 min and in 10 mM ACh for 30 min ( B ) , or in buffer for 15 min and 10 mM phenyltrimethylammonium for 30 min (C). The receptor was reacted with 100 I . ~ M EPCD in the continued presence of the binding site ligands and handled further just as in Fig. 1B. Tritium counts loaded and recovered were: A, 8280,6549; B, 4122,5966; C, 4875,6304.2-pl aliquots were taken to determine input.

3% ( n = 10) of the initially disulfide-linked ["HIGC was amide-coupled. After reduction and electrophoresis of the reacted receptor, 46+3% ( n = 11) of the amide-coupled ["HI GC loaded on the gels was recovered in monomeric receptor subunits.

We relied on the characteristic electrophoretic mobilities of the receptor subunits (Weill et dl., 1974) to identify them on stained gels and to associate radioactivity in the gel slices with the subunits. To verify our identification of the subunits after the cross-linking procedure, we used anti-subunit monoclonal antibodies (Fig. 3). Anti-a and anti-6 antibodies each bound to a single band with the expected apparent molecular weight. The binding of anti+ and anti-y monoclonal antibodies indicated partial degradation of these subunits, and a significant amount of anti-y staining overlapped anti-P staining.

The major site of EPCD-induced amide-coupling of disulfide-linked ['HH]GC was 6 (Fig. 1B). 6 had its usual mobility and did not overlap with other subunits (Fig. 3). In four independent experiments, the fraction of amide-coupled ["HI GC recovered in each monomeric subunit relative to the total recovered in all of the monomeric subunits was 21 f 1% in a, 15 & 2% in p, 28 f 2% in y, and 37 + 2% in 6. In making these calculations, we assigned the radioactivity in slices 100- 109 to y and that in slices 110-117 to /3 (Fig. lB) , consistent with the immunoblotting results (Fig. 3).

The presence during the EPCD reaction of 10 mM acetylcholine or 10 mM phenyltrimethylammonium inhibited the amide coupling of ['HIGC to 6 by 71 & 2% or 54 & 2%, respectively (Fig. 4). The incorporation into p and y was also somewhat decreased in the presence of agonists.

Subunits Cross-linked by ['HJGC-Only a and 6 had enough disulfide-linked ['HIGC to be the origin for most of ["HIGC subsequently amide-coupled to 6 (Fig. 1, A and B ) . To determine whether a or 6 was the predominant origin, we decreased the initial disulfide-linking of ["HIGC to a and increased it to 6 by a two-step procedure. First we reduced and alkylated a large fraction of a Cysl"'/Cysl":', and second we completely reduced the 6-6 disulfide with 2 mM DTT. This procedure decreased the disulfide linking of ["HIGC to a by factors of 2 to 7 and increased disulfide-linking of [''HIGC to 6 by a factor of 2, compared to controls (Fig. 1, C and A, and Table 111). Despite the increase in ["HJGC disulfide-linked to delta, the amount of ['HIGC subsequently amide-linked to delta decreased by a factor of 2 compared to controls (compare Fig. l , D and B ) ; this decrease in ["HIGC amide-coupled to 6 corre- lated with the decrease in ["HIGC disulfide-linked to a. a, therefore, appears to be the predominant origin of ["HIGC subsequently amide-coupled to 6.

We quantitated the above conclusion. We estimated the fractions of ['HIGC amide-coupled to 6 that originated in a and 6. A priori, ['HIGC disulfide-linked to any one subunit could be amide-coupled by EPCD to the same subunit or to any of the other subunits. We assumed that there are cross- linking factors that express the efficiency of these amide- coupling reactions. We also assumed initially that the amount of [:%H]GC amide-coupled to a and 6 originating in p and y was small compared to that originating in a and 6. We needed therefore to determine the cross-linking factors for a to a (A1), for a to 6 (A4), for 6 to a (Dl), and for6 to 6 (D4). These were determined as variables in two sets of simultaneous equations (Table 111, Footnote c).

We experimentally manipulated the amounts of ['HIGC disulfide-linked to a and 6 and thus of ['HIGC subsequently amide-linked to a and 6 in two ways. One was to alkylate a Cys1""/Cys1":' with NEM and to increase the reduction of 6, as


1

2

3

4

5

6

7

8

9

10

11

12

6 Subunit Carboxyl Close to A C h Receptor Binding Site Residues 1- 58: MW= 6716 . . . . . . . . . . . V N ' E E E R L I N D L L I V I A I B L T L S N L J S L K E T D E T L T S N V W M

Residues 59- 160: MW= 12012

D ~ ~ D ~ ~ ~ E Y S D I S I L R L e P E L V W I P D ~ ~ ~ ~ A Y P - ~ N G ~ TWLPPAI[SCPINVLYPPPMIQNCSLKPPALWYDANEITll

. . . . . . . . . . . .

Residues 161- 163: MW= 377

DIM

Residues 164- 257: Hw= 10949

TDTIDGKDYPIEWIIIDP~GEUEIIHlZPARKNIYPDKFPNGTNYQDVITYLIIRR KPLFYVINFITPCVLlSPLASLb.FYLP&ESGEIM_

. . . . . . . . . . . .

Residues 258- 293: MW= 3843 . . . . . . . STAISVLIAOAWLLLTSQRLPETALAVPLIG~

Residues 294- 296: m- 410

En! Residues 297- 340: MW= 5016

SLVTGVIVNCGI~PRTPSTHVLSTRVKQIPIJXLPRIIJW . . . . . . . .

Residues 341- 382: MW= 4950 . . . . . . . . S ~ E S E Q P M I Q N D ~ S ~ G Y I S ~ Q E Y P N I K S R S

Residues 383- 458: MW= 8629 . . . . . . P E K Q ~ E R H G ~ ~ ~ I G P I ~ ~ ~ E I ~ G I ~ T N Y I V K ~ I ~ ~ A Y D E E VGNWNLVGQTID-

Residues 459- 465: MW= 820

l!J.m?a Residues 466- 475: MW= 1139

VLGTIPIPVU

Residues 476- 501: MW= 2903

GHPNAPPAKPPEGDPPDYSSDHPRCA

. .

. . . . . SCHEME 2. Cyanogen bromide cleavage fragments of the 6 subunit.

The predicted membrane-spanning segments are underlined.

I 'I 1 [ 200 9

4 0 20 40 60 80

ELUTION TIME (rnin)

FIG. 5. HPLC of ["HIGC amide-coupled to the d subunit.. Receptor (28-nmol binding sites) was mildly reduced, reacted with ["HIGCP and EPCD, exhaustively reduced, and electrophoresed, just as in Fig. lR, except that the sample was loaded for electrophoresis in one large well. The band containing the 6 subunit was cut out, and d was electroeluted, dried, dissolved by adding 100 pl of water, precipitated with 90% acetone, dried, and dissolved and cleaved in 100 pl of 1 id CNBr in 80% TFA for 12 h a t room temperature (DiPaolo et al., 1989). We added 100 pl of water to the cleavage mixture, dried it, and dissolved it in 70% TFA. This was injected onto a Vydac C4, 25- X 0.46-cm column, and eluted with a mixture of 0.07% TFA (buffer A) and 60% acetonitrile, 40% isopropanol, 0.03% TFA (buffer B), as follows: 0-5 min, 15% B; 5-34 min, 15- 35% B; 34-59 min, 35-60% B; 59-74 min, 60-90% B; 74-79 min, 90% B; and 79-80 min, 90-15% B. Input of tritium was approximately 14,600 cpm (1-p1 aliquot); 17,300 counts were recovered. The flow rate was 1 ml/min, and 1 min fractions were collected. The tritium in 100-pl aliquots is plotted.

described in Fig. 1, and another was to inhibit with ACh both the reduction of a C y ~ ' ~ ' / C y s ' ~ ~ and their reaction with ['HI GCP (Table 111). Each experiment yielded two sets of data, one obtained under control conditions and another under blocked conditions, and these data were used to solve the simultaneous equations for the cross-linking factors.

22609

L.. 1 5

0 20 4 0 R O

SLICK NtiMilER

FIG. 6. Apparent molecular weight of the CNBr fragment of 6 containing the majority of amide-coupled ['HIGC. Fraction 58 from a HPLC run duplicating that shown in Fig. 5 was dried, dissolved in Laemmli sample buffer, and loaded in two lanes of a slab gel. This gel consisted of three layers, 4, 10, and 16.5% acrylamide (Schagger and von Jagow, 1987), respectively. After electrophoresis, one sample lane and one standard lane were electroblott,ed onto polyvinylidene difluoride paper, stained, and photographed ( A ). Lane 1, myoglobin fragments (Sigma SDS-17) with molecular weights of 16,950 ( a ) , 14,440 (b ) , 10,600 ( c ) , 8,160 ( d ) , and 6,210 ( e ) . Lane 2, fraction 58 from HPLC shown in Fig. 5. The arrowhead identifies the major ['HIGC-labeled fragment with an apparent molecular weight of 15,700. The other sample lane was stained, sliced, and counted ( R , circles). Myoglobin fragments and cu-bungarotoxin (M, 8,250) were run in parallel as molecular weight standards (triangles). The input of tritium was 3000 counts per min; 3,200 counts per min were recovered.

For our conclusion that most of ['HJGC amide-coupled to 6 originated in a, A, is the most important factor. To calculate the fraction of ['HIGC amide-coupled to 6 that originated in a, we multiplied the amount of ["HIGC disulfide-linked to a (ALPHA in Table 111) by A4 and divided by the total ["HIGC amide-coupled to delta (DELTA' in Table 111). The result of five experiments was that 91 k 9% of the ["HJGC amide- coupled to 6 originated in a (Table 111). We can also estimate that ["HIGC disulfide-linked to a was three times (A4/AI) more likely to be amide-coupled to 6 than to a itself.

We tested our assumption that p and y were not the origins of significant ['HIGC cross-links to 6. We assumed instead that 5.5% of ['HIGC initially in p and y was subsequently amide-coupled to 6; i.e. the same fraction of ["HIGC disulfide- linked to a that was amide-coupled to 6. We solved the simultaneous equations again, now including these hypothet- ical contributions from p and y. The result was that the cross- linking factor from N to 6, A4, was 5.4 * 2%, only slightly different from the previously calculated value, and N was still the origin of 88 k 12% of the ['HIGC amide-coupled to 6. Furthermore, the cross-linking factors from ,B and y to 6 could not be larger than 5.5%, because larger factors resulted in negative factors for the cross-linking of ["HIGC from 6 to 6. Therefore, even if there were some cross-linking from p and y to 6, about 90% of ["HIGC amide-coupled to 6 would still have originated in a.


TABLE IV N-terminal sequences of the major L3H]GC-labeled CNBr fragments of 6

The major band from a polyacrylamide gel, which was a duplicate of the one described in Fig. 6, was blotted onto polyvinylidene difluoride paper as described by Matsudaira (1987) and sequenced by automated Edman degradation.

Cycle Residue

D L M T D T I D G K D Y P I E W I I I D P E A 1 20" 22 2 8 3 4 16 9 5 25 25 6 10 7 7 5 4 8 16 9

10 11 12 13 14 2 4 15

- 4

16 17 18 19 2 2 20

1 -

20 2 36

16 4 3

2 4 6 3 3

2 2 3 3 3 -

4 2 4 4 2 - 4 4

- 3 3 3 2 2 2 2

1 2 The phenylthiohydantoin derivatives of the amino acid residues released in each cycle of automated Edman

degradation are expressed in picomoles.

The Region of 6 Amide-linked to PHIGC-6 subunit amide- coupled to [3H]GC was cleaved with CNBr (see Scheme 2), and the fragments were separated by HPLC (Fig. 5). Most of the radioactivity eluted around 58 min and coeluted with a UV-absorbing peptide. Automated Edman degradation of fraction 58 yielded the N-terminal sequences of CNBr fragment 1 and CNBr fragment 4 (data not shown). Fraction 58 was also subjected to SDS-PAGE in a system that resolves low molecular weight peptides (Fig. 6). Three Coomassie Brilliant Blue-stained bands of apparent molecular weights 15,700, 37,000, and 42,000 were observed (Fig. 6 A ) . The 15,700-dalton component corresponded to the major peak of radioactivity in the gel slices (Fig. 6B). The higher molecular weight doublet corresponded to two minor peaks of radioactivity and had the N-terminal sequence of CNBr 1. The major peak of radioactivity from the gel showed two coherent N- terminal sequences; one starting from Asp1", at the beginning of the tripeptide CNBr 3 (Scheme 2) and continuing through the first 17 residues of CNBr 4, and the second starting at ThP4, at the beginning of CNBr 4 (Table IV). These two sequences were consistent with incomplete cleavage of the Met-Thr bond between CNBr 3 and CNBr 4. (Also, incomplete Edman degradation at resulted in two minor sequences, parallel to the major sequences but retarded by one cycle.)

Because of the length of CNBr 4 and the incomplete cleavage between CNBr 3 and CNBr 4, we have not identified the labeled residue(s). We observed repeatedly, however, no re- lease of radioactivity in the first cycle of Edman degradation. Therefore, Asp'61, the first residue of CNBr 3, was not labeled by [3H]GC. After 30 cycles of cleavage we recovered 40-50% of the radioactivity loaded on the filter; 20-30% was retained on the filter. We conclude that the Asp and/or Glu residues to which [3H]GC was amide-coupled were in CNBr 4 (6 164- 257) and that most if not all these Asp and/or Glu were among the first 30 residues of CNBr 4.

DISCUSSION

The use of bifunctional cross-linking agents to probe protein structure has been widespread (Peters and Richards,

1977), and cross-linking agents containing the dithiopyridyl functional group have been described ( e g . Chong and Hodges, 1981; Dhanasekaran et al., 1988). [3H]GCP has some useful features. It is readily synthesized with radioactive glycine. It can be used to cross-link between sulfhydryls and carboxyls in two steps (Scheme 1): The first is disulfide bond formation, and the second is amide bond formation following the acti- vation of carboxyls with a carbodiimide. Because the first reaction is reversed by reduction, it is possible to map sepa- rately the two sites of reaction.

Because of the unusual susceptibility to reduction of the disulfide at the ACh binding site (Karlin, 1969; Kao and Karlin, 1986), we could direct [3H]GCP mostly (82%) to this site. The disulfide-linking of [3H]GC to a was blocked by agonists, which inhibit the reduction of the binding site disulfide (Damle and Karlin, 1980), and by affinity labels of the binding site disulfide (Karlin, 1969; Weill et al., 1974; Damle et al., 1978). The binding site disulfide is formed by Cys"* and Cyslg3 (Kao et al., 1984; Kao and Karlin, 1986). By peptide mapping, we found that all [3H]GC in a was in CNBr 4 (Table I1 and Fig. 2B), which contains Cy~ ' '~ /Cys '~~ and no other Cys residues. All of this evidence points to as the unique residues in a reacting with [3H]GCP. Under the conditions we used, approximately 70% of the a subunits were disulfide-linked to [3H]GC (5 nmol of [3H]GC in a per mg of receptor compared to 7 nmol of a per mg of receptor).

Disulfide-linked [3H]GC was amide-coupled to the receptor by EPCD. Under conditions that resulted in approximately 50% recovery of monomeric subunits, about 18% of the initially disulfide-linked [3H]GC was amide-coupled to the receptor. The exent of amide-coupling was determined in all cases as the radioactivity remaining associated with the receptor after exhaustive reduction in SDS. Although 82% of the disulfide-linked [3H]GC was initially associated with a, after EPCD the largest fraction (37%) of the amide-coupled [3H] GC was associated with 6. We estimated that about 90% of the [3H]GC amide-coupled to 6 originated in a. Thus, a region of &containing carboxyl groups is within 0.9 nm of a Cyslg2/ Cys"3.

6 Subunit Carboxyl Close to ACh Receptor Binding Site 22611

[3H]GC was also amide-coupled to the other subunits. We calculated that 61% of [3H]GC amide-coupled to a originated in a (Table 111). Thus, a contains carboxyls within 0.9 nm of a C y ~ ' ~ ~ / C y s ' ~ ~ . However, our mapping of these carboxyls would be complicated since 40% of [3H]GC amide-coupled to a originates in 6 and not in a C y ~ ' ~ ~ / C y s ' ~ ~ . p and y were also amide-coupled to [3H]GC, y to a greater extent than either a or 0. The partial degradation of p and y during the long cross- linking procedure and their subsequent overlapping on SDS- PAGE complicated our analysis of these subunits. Thus, we focused on 6 , which was not degraded and was the major site of amide-coupling of [3H]GC.

We were unable, however, to detect a unique radioactive a- 6 dimer by either one- or two-dimensional SDS-PAGE. Recep- tor cross-linked by [3H]GC, but not subjected to a final reduction, gave (on SDS-PAGE) a smear of radioactivity in the molecular weight range between 6 monomer and the top of the gel and multiple bands of immunoreactivity to anti-a and to anti-6 monoclonal antibodies (not shown). This profusion of species reflected the variety of directly cross-linked and [3H]GC-cross-linked subunits. We would expect that the t3H] GC-cross-linked a-6 dimer would constitute only 2' of the total covalently bound [3H]GC on the gel, and even this 2% would be split between a cro~s-linkedby[~H]GC to monomeric 6 and a cross-linked to disulfide-linked 6-6 dimer. Only after reduction of all disulfides could we sort out this mixture. After the final reduction, about 50% of amide-coupled [3H]GC was associated with monomeric subunits, and our analysis was based on this 50%. It seems unlikely that the subunits that were directly cross-linked by EPCD would have reacted dif- ferently with [3H]GC than those subunits that were not directly cross-linked. Even had there been differences, however, our conclusion that [3H]GC cross-linked the ACh binding site residues, (Y C y ~ ' ~ ~ / C y s ' ~ ~ , to residues on the 6 subunit, would not be affected. At worst, we might be underestimating the crosslinking of other subunits.

Based on atomic models, we estimated that the maximal distance bridged by 3H-GC is 0.9 nm. This is also the maximal length of cholinoxycarbonylmethyl, the moiety added to a C y ~ ' ~ ~ / C y s ' ~ ~ by the reaction of the affinity label BAC with reduced receptor. BAC is an irreversible activator of the reduced receptor (Silman and Karlin, 1969), and the quaternary ammonium group in BAC and in ACh are very likely interacting with the same negative subsite, at most 0.9 nm away from the binding site disulfide (Karlin, 1969). The binding site disulfide is formed by CYS'~' and Cyslg3 (Kao and Karlin, 1986), which are affinity labeled by [3H]MBTA (Kao et al., 1984). [3H]BAC labels a in reduced receptor with high specificity, and the reaction of [3H]BAC and that of [3H] MBTA are mutually exclusive (Damle et al., 1978). Further- more, there is no readily reducible disulfide in a other than that formed by Cy~ '~ ' /Cys '~~ (Kao and Karlin, 1986). There- fore, BAC also reacts with C y ~ ' ~ ~ / C y s ' ~ ~ . Thus, there is a negative subsite of the ACh binding site that is at most 0.9 nm from a C y ~ ' ~ ~ / C y s ' ~ ~ , when the binding site is occupied by ACh or a similar agonist.

The carboxyl(s) on 6 that are amide-coupled to [3H]GC are likely to contribute to this negative subsite. The distance from these 6 carboxyls to a C y ~ ' ~ ~ / C y s ' ~ ~ is 0.9 nm or less, corresponding to the maximal distance from the negative subsite to Cy~'~'/Cys'~~. In addition, the EPCD-induced reaction of

'82% of all disulfide-linked [3H]GC was in a, 18% of disulfide- linked [3H]GC became amide-coupled, 46% of amide-coupled [3H]GC was recovered in monomeric subunits, 37% of this was in 6, and 90% of ['HJGC amide-coupled to 6 originated in a: 82 X 18 X 46 X 37 x 90 = 2%.

disulfide-linked [3H]GC with these carboxyls was slowed in the presence of agonists (Fig. 4). High concentrations of agonist were required to inhibit the EPCD reaction, consistent with our observation that mild reduction of the receptor and its reaction with ACP inhibited the binding of [3H]ACh (data not shown) and with previous observations that mild reduction alone decreased the affinity of the receptor for agonists (Karlin and Bartels, 1966; Schiebler et al., 1977).

We do not presume that the primary ammonium group of GC has appreciable affinity for the negative subsite; this site strongly prefers quaternary ammonium groups or charged nitrogens otherwise surrounded by hydrophobic groups (Spi- vak and Albuquerque, 1982). We do assume, however, that the effective concentration of GC tethered by a disulfide bond to C y ~ ' ~ ~ / C y s ' ~ ~ is very high within a radius of 0.9 nm of this disulfide and that the ammonium of tethered GC is likely to react with an accessible, EPCD-activated carboxyl within this radius.

We cleaved [3H]GC-labeled 6 with CNBr and isolated the labeled components by reversed-phase HPLC and by SDS- PAGE. The predominantly labeled band had an apparent molecular weight of 15,700 and was a mixture of fragments, one starting at and another at Thr"j4. The Met-Thr bond is resistant to CNBr cleavage (Schroeder et al., 1969), and despite our use of CNBr in TFA (Tarr, 1986; DiPaola et al., 1990), the bond between Met'63 and T h P 4 , between CNBr 3 and CNBr 4, was incompletely cleaved. The predicted molecular weight of CNBr 4 is 10,949 plus about 2000-3000 for the N-linked oligosaccharide attached to Asdo' (Nomoto et al., 1986), or about 13,000-14,000. The predicted molecular weight of the fusion of CNBr 3 and CNBr 4 is 378 more than that of CNBr 4. Since the next fragment, CNBr 5 has a molecular weight of 3,842, it is unlikely that the major labeled fragments continued beyond the end of CNBr 4. Only the first 61 residues of CNBr 4 are predicted to be extracellular (Scheme 2; Noda et al., 1983).

The extracellular part of the Torpedo 6 CNBr 4 has a net negative charge since it contains 11 Asp or Glu residues and only 9 Lys or Arg or His residues. More striking is the electronegativity of the first 30 residues of CNBr 4, which has a net charge of minus 8. This region also contains 4 Asp or Glu residues that are conserved in the aligned sequences of 6 and y subunits from five species and in epsilon subunits from two species. These correspond to Torpedo 6 Asp'", G1ul%, and Glu'". Most of the radioactivity recovered by us during sequencing of CNBr 4 was in the first 30 cycles of Edman degradation. Because of the length of CNBr 4 and the incomplete cleavage between CNBr 3 and CNBr 4, further cleavage is necessary for us to identify the labeled residues unambiguously.

Our conclusions are consistent with previous work. There are two ACh binding sites per receptor monomer (Reynolds and Karlin, 1978), and these two sites are intrinsically different (Damle and Karlin, 1978; Damle et al., 1978; Neubig and Cohen, 1979; Sine and Taylor, 1981; Dowding and Hall, 1987). The subunit composition of the receptor is aZ-p-y-6 (Reynolds and Karlin, 1978), and the order of the subunits around the central channel is a-y-a-p-6 (Karlin et al., 1983). Based on the expression of incomplete combinations of receptor subunits, Blount and Merlie (1989) inferred that a and y organize one ACh binding site and that a and 6 organize another, consistent with the above order of the subunits. Furthermore, the a/6 site had higher affinity for agonist and lower affinity for competitive antagonist than the a / y site. That these sites might be at the interface between subunits was suggested by the photoincorporation of the competitive antagonist d-tub-


ocurarine into y and 6, as well as into a (Pedersen and Cohen, 1990). Our previous and current results indicate that one ACh binding site is formed between the region around a Cyslg2/ CYS’’~ and residues within 6 164-224. It is very likely that the other ACh binding site is formed between the correponding regions in the second a and in y.

Acknowledgments-We thank Dr. Michael Zigorsky for NMR analysis of ACP, Dr. Pat Warne for mass spectrometry of ACP, Dr. Jon Lindstrom for monoclonal antibodies, Dr. Arnold Ruoho and Dr. Ed Fujimoto for valuable discussions, Drs. David Stauffer, Jonathan Javitch, and Myles Akabas for helpful comments on the manuscript, and Gilda Salazar-Jimenez for expert technical assistance.

REFERENCES

Abramson, S. N., Li, Y., Culver, P., Taylor P. (1989) J. Biol. Chem.

Boulter, J., O’Shea-Greenfield, A., Duvoisin, R. M., Connolly, J. G., Wada, E., Jensen, A., Gardner, P. D., Ballivet, E. S., Deneris, E. S., McKinnon, D., Heinemann, S., and Patrick, J. (1990) J. Biol. Chem. 265,4472-4482

264,12666-12672

Blount, P., and Merlie, J. P. (1989) Neuron 3, 349-357 Brocklehurst, K., and Little, G. (1973) Biochem. J . 133, 67-80 Chong, P. C. S., and Hodges, R. S. (1981) J. Biol. Chem. 256, 5064-

5070 Claudio, T. (1989) in Frontiers in Molecular Biology: Molecular Neu-

robiology (Glover, D. M., and Hames, B. D., eds) pp. 63-142, IRL Press, Oxford

Czajkowski, C., DiPaolo, M., Bodkin, M., Salazar-Jimenez, G., Holtz- man, E., and Karlin, A. (1989) Arch. Biochem. Biophys. 272,412- 420

Damle, V. N., McLaughlin, M., and Karlin, A. (1978) Biochem. Biophys. Res. Commun. 84,845-851

Damle, V. N., and Karlin, A. (1978) Biochemistry 17, 2039-2045 Damle, V. N., and Karlin, A. (1980) Biochemistry 19, 3924-3932 Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth,

C., Chang, J.-Y., Lazure, C., Chretien, M., and Changeux, J.-P.

Dhanasekaran, N., Wessling-Resnick, M., Kelleher, D. J., Johnson, G. L., and Ruoho, A. E. (1988) J. Bid. Chem. 263,17942-17950

DiPaolo, M., Czajkowski, C., and Karlin, A. (1989) J. Biol. Chem.

DiPaolo, M., Kao, P. N., and Karlin, A. (1990) J. Bid. Chem. 265,

Dougherty, D. A,, and Stauffer, D. A. (1990) Science 250, 1558-1560 Dowding, A. J., and Hall, Z. W. (1987) Biochemistry 26,6372-6381 Galzi, J.-L., Revah, F., Black, D., Goeldner, M., Hirth, C., and

Galzi, J.-L., Revah, F., Bessis, A., and Changeux, J.-P. (1991) Annu.

(1988) Biochemistry 27, 2346-2357

264,15457-15463

11017-11029

Changeux, J.-P. (1990) J. Bid. Chem. 265,10430-10437

Reu. Pharmacol. 31, 37-72

Hamilton, S. L., McLaughlin, M., and Karlin, A. (1979) Biochemistry

Kao, P. N., Dwork, A. J., Kaldany, R.-R. J., Silver, M. L., Wideman, J., Stein, S., and Karlin, A. (1984) J. Biol. Chem. 259, 11662- 11665

18,155-163

Kao, P. N., and Karlin, A. (1986) J. Biol. Chem. 261,8085-8088 Karlin, A. (1969) J. Gen. Physiol. 54 (suppl.), 245-264 Karlin, A. (1977) Methods Enzymol. 46, 582-590 Karlin, A. (1991) Harvey Lect. 85, 71-107 Karlin, A., and Bartels, E. (1966) Biochim. Biophys. Acta 126, 525-

535 Karlin, A., Holtzman, E., Yodh, N., Lobel, P., Wall, J., and Hainfeld, J. (1983) J. Bid. Chem. 258, 6678-6681

Laemmli, U. K. (1970) Nature 227,680-685 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)

Matsudaira, P. (1987) J. Bid. Chem. 262, 10035-10038 Means, G. E., and Feeney, R. E. (1971) Chemical Modification of

Neubig, R. R., and Cohen, J. B. (1979) Biochemistry 18,5464-5475 Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S.,

Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983b) Nature 302, 528-532

Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y., and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242

Pedersen, S. E., and Cohen, J . B. (1990) Proc. Natl. Acad. Sci. U. S.

Peters, K., and Richards, F. M. (1977) Annu. Reu. Biochem. 46,523-

Reynolds, J. A,, and Karlin, A. (1978) Biochemistry 17, 2035-2038 Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-

Schiebler, W., and Lauffer, L., and Hucho, F. (1977) FEBS Lett . 81,

Schoepfer, R., Conroy, W. G., Whiting, P., Gore, M., and Lindstrom,

Schroeder, W. A., Shelton, J. B., and Shelton, J. R. (1969) Arch.

Silman, H. I., and Karlin, A. (1969) Science 164, 1420-1421 Sine, S. M., and Taylor, P. (1981) J. Bid. Chem. 256,6692-6699 Smith, I. (1969) Chromotogruphic and Electrophoretic Techniques.

Vol. I , p. 122, John Wiley & Sons, New York Spivak, C. E., and Albuquerque, E. X. (1982) in Progress in Cholin-

ergic Biology: Model Cholinergic Synapses (Hanin, I., and Goldberg, A. M, eds) pp. 323-357, Raven Press, New York

Tarr, G. E. (1986) in Methods of Protein Microcharacterization

Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. (Shively, J. E., ed) p. 164, Humana Press, Clifton, NJ

U. S. A. 76,4350-4354 Tzartos, S., Langeberg, L., Hochswender, S., Swanson, L., and Lind-

Weill, C. L., McNamee, M. G., and Karlin, A. (1974) Biochem. strom, J. (1986) J. Immunol. 10, 235-253

Biophys. Res. Commun. 61,997-1003

J. Bid. Chem. 193, 265-275

Proteins, pp. 144-147, Holden-Day, Inc., San Francisco

A. 87,2785-2789

551

379

39-42

J . (1990) Neuron 5,35-48

Biochem. Biophys. 130,551-556

Documents

Agonist Binding Site of Torpedo Electric Tissue Nicotinic