differential surface accessibility of α(187–199) in the torpedo acetylcholine receptor α...

Article No. mb982001 J. Mol. Biol. (1998) 282, 317±330

Differential Surface Accessibility of aaa(187±199) in theTorpedo Acetylcholine Receptor aaa Subunits

Robert H. Fairclough*, George M. Twaddle, Eswari Gudipati, Mike Y. Linand David P. Richman

University of CaliforniaDavis, Department ofNeurology, Davis, CA 95616USA

E-mail address of the corresponrhfairclough@ucdavis.edu

Abbreviations used: ACh, acetyla-Btx, a-bungarotoxin; BAC, brombovine serum albumin; DMF, dimd-tubocurarine; DTT, dithiothreitoldimethylaminopropyl)carbodiimidlinked immunosorbant assay; Fab,fragment of an antibody; F-Btx, calabeled bungarotoxin; F-ACh recepcadaverine-labeled acetylcholine recarboxy¯uorescein-labeled monoclHepes-buffered saline; mAb, monoMBTA, 4-(N-maleimido)benzyltrimiodide; PBS, phosphate-buffered sa1 psi � 6.9 kPa; R-Btx carboxytetrarhodamine-labeled bungarotoxin; Rcarboxytetramethylrhodamine-labesulfo-N-hydroxysuccinimide; SPR,resonance; TFA, tri¯uoroacetic acid

0022±2836/98/370317±14 $30.00/0

We have probed the surface accessibility of residues a187 to a199 of theTorpedo acetylcholine receptor with monoclonal antibody 383C, whichbinds uniquely to these residues. However, 383C binds to only one of thetwo a subunits in the membrane-bound receptor, neither of the two sub-units in carbamylcholine-desensitized receptor, and to both a subunits inTriton X-100 solubilized receptor. The kinetics of association and dissoci-ation of 383C with the peptide a(183±199) compared to those with themembrane-bound receptor suggest that all but a single hydrogen bond ofaf®nity derives from contacts between this peptide and the monoclonalantibody paratope. Inhibition of 383C binding by a-bungarotoxin selec-tively directed to the a subunit correlated with the high-af®nity d-tubo-curarine binding site, along with a lack of inhibition by a-bungarotoxindirected to the a subunit correlated with the low-af®nity d-tubocurarinebinding site, suggests that the 383C epitope on the membrane-boundreceptor resides on the a subunit associated with the high-af®nity d-tubo-curarine binding site. The results presented here suggest a structuralbasis for the differences between the two receptor acetylcholine bindingsites.

# 1998 Academic Press

Keywords: acetylcholine receptor; anti-acetylcholine receptor monoclonalantibody; acetylcholine binding sites; a-bungarotoxin binding sites;d-tubocurarine binding sites

Introduction

Of the Torpedo acetylcholine (ACh) receptor sub-units, the two a subunits play a central role in

ding author:

choline;oacetylcholine; BSA,ethylformamide; dTc,; EDC, ethyl-3-(3-e; ELISA, enzyme-antigen-binding

rboxy¯uorescein-tor, ¯uoresceinceptor; F-mAb,onal antibody; HBS,clonal antibody;ethylammoniumline; psi,methyl--mAb,

led mAb; SNHS,surface plasmon.

binding the two equivalents of ACh required forchannel activation. Furthermore, each a subunit isassociated with one of two competitive antagonistbinding sites (Blount & Merlie, 1989; Pedersen &Cohen, 1990) as well as one of two snake a-neuro-toxin binding sites. A part of these binding sitesappears to involve a common set of residues in thesame linear peptide sequence of each a subunit;residues 185 to 200 (Middleton & Cohen, 1991).However, considerable evidence has accumulateddocumenting differential positioning and chemicalreactivity of the amino acid residues in this peptidedepending upon the a subunit in which theyreside.

Initially, investigators reported that the AChreceptor covalently incorporates either 4-(N-maleimido)benzyltri[3H]methylammonium iodide(MBTA; Damle & Karlin, 1978) or bromoacetylcho-line (BAC; Moore & Raftery, 1979) following mildreduction with DTT at only half the number ofsites that bind a-neurotoxins. Each reagent incor-porates selectively into the a subunit at C-192/193,suggesting a difference in the chemical reactivity of

# 1998 Academic Press

318 Differential Surface Accessibility of �(187±199)

a disul®de bond in the two a subunits. Using BAC,Wolosin et al. (1980) demonstrated that two siteson Torpedo ACh receptor can be labeled in Ringer'ssolution at and above 23�C using higher concen-trations of BAC than are required for labeling onlythe ®rst site. In studying this differential reactivityto BAC, Walker et al. (1984) found that the secondsite seems to rapidly reoxidize, rendering it inac-tive to modi®cation with BAC if reducing agent isremoved.

In the closed resting state, the ACh receptorexhibits two widely different af®nities for agonist:5 mM and >3.5 mM for mouse ACh receptor(Jackson, 1988); and 4.2 mM and 400 mM for Torpe-do ACh receptor expressed in ®broblasts (Sine et al.,1990). Furthermore, in equilibrium binding exper-iments with the competitive antagonist d-tubocur-arine (dTc), Neubig & Cohen (1979) havedistinguished two dTc sites on the Torpedo AChreceptor: a high-af®nity site with Kd 33 nM and alow-af®nity site with Kd 7.7 mM. Ratnam et al.(1986) correlated the ®rst site modi®ed by BACwith the high-af®nity dTc binding site. Cohen alsocites data from his laboratory indicating thatMBTA preferentially labels the a subunit associ-ated with the high-af®nity dTc binding site(Pedersen et al., 1986; Dreyer, 1984). Blount &Merlie (1989) correlated development of high-af®-nity and low-af®nity dTc binding with coexpres-sion in QT-6 ®broblasts of mouse a and g subunits,and a and d subunits, respectively. Likewise Sine& Claudio (1991) found high-af®nity dTc bindingto mouse a2bg2 expressed in 3T3 cells and low-af®-nity binding to a2bd2. Using photoaf®nity labelingLangenbuch-Cachat et al. (1988) observed speci®cphoto-incorporation of p-(dimethylamino)benzene-diazonium ¯uoroborate, a reversible competitiveantagonist, into the a and the g subunit. Thephoto-incorporation into both subunits wasreduced by 10 mM dTc and 100 mM carbamylcho-line. In addition, Pedersen & Cohen (1990) foundphoto-incorporation of [3H]dTc into the a and gsubunits of the ACh receptor at lower concen-trations of antagonist than for photo-incorporationinto the d subunit. Chiara & Cohen (1997) sub-sequently identi®ed aY190, aC192, aY198, gW55and dW57 to be the major sites of speci®c dTcincorporation. These data are consistent with theproposition that the high-af®nity dTc binding sitelies near the subunit interface between a and g,and that two primary ligand binding sites of theACh receptor lie near the interfaces of the a sub-units with the g subunit and the d subunit, respect-ively. In support of this, Czajkowski & Karlin(1991) have identi®ed a carboxylate residue on thed-subunit 0.9 nm from a-Cys192±193. In addition,Dunn et al. (1993) report a BAC-reactive site gener-ated by sodium borohydride reduction of the AChreceptor that results in labeling of both the a and gsubunits. This dual labeling is blocked by priorreaction of the ACh receptor a subunit with DTTand BAC, suggesting that the BAC site is near thea±g interface.

In contrast to this interface model, Unwin (1996)has suggested that acetylcholine binds in pocketslocated within the two a subunits, and the differ-ences in chemical reactivity and ligand af®nityresult from non-equivalent conformations of the asubunits.

With respect to the two a-neurotoxin bindingregions, Conti-Tronconi et al. (1990) characterized areversible a-bungarotoxin (a-Btx) site and an irre-versible a-Btx site in Torpedo ACh receptor-enriched membranes. The reversible site convertsto an irreversible site in Triton X-100. Conti-Tronconi et al. (1990) further suggest a role forcarbohydrate in distinguishing these two sites.Dunn et al. (1993) found that extensive BAC modi-®cation of the ACh receptor completely inhibits thebinding of a-Btx to membrane-bound ACh receptorand 50% of the binding of a-Btx to detergent-solu-bilized receptor, results that suggest that BACmodi®cation alters the a-Btx binding to the irre-versible site on membrane-bound ACh receptor.

To study the differences between the two siteslocalized on unique a subunits, Dowding & Hall(1987) have produced a panel of anti-ACh receptormonoclonal antibodies (mAbs) that block 50% ofthe a-Btx binding to Tween 80-solubilized TorpedoACh receptor. The antibodies in this panel fall intoone of two groups that seem to inhibit binding ofa-Btx to two different sites on the same ACh recep-tor. One group, A, inhibits the binding of a-Btx toa site characterized kinetically by an initial associ-ation rate �106 Mÿ1sÿ1 and the other group, B,inhibits binding of the toxin to a site characterizedby an initial rate �105 Mÿ1 sÿ1. Each mAb in groupA, together with any one of the mAbs in group B,can completely inhibit the binding of a-Btx to theACh receptor.

Fels et al. (1986) have also raised and character-ized anti-ACh receptor mAbs that are blocked bya-Btx and bind 1:1 to detergent-solubilized AChreceptor. In this class is mAb WF6, which by itselfcompletely inhibits a-Btx binding but inhibits ago-nist binding at only one of the two sites.

To probe the local environment of the twoacetylcholine binding sites, we have employedmonoclonal antibody 383C from an anti-AChreceptor library prepared by Gomez & Richman(1983). This anti-ACh receptor mAb was initiallytargeted for interest because of the ability of a-Btxto inhibit its binding to the ACh receptor. We havepreviously mapped the epitope of this mAb to thea subunit sequence residues 187 to 199 and to themembrane-bound ACh receptor a2 subunit 6�clockwise from the a2 vertex and 35 AÊ up fromthe level of the phosphate head groups of the lipidbilayer (Fairclough et al., 1998a). Here, we presentstudies characterizing the binding properties ofthis mAb in the presence and absence of otherACh receptor ligands. The results of this study pro-vide insights into the structural differencesbetween the two acetylcholine binding sites on theACh receptor, a correlation between the function-ally de®ned high and low-af®nity dTc sites, and

Table 1. Stoichiometry of a-bungarotoxin and mAbbinding to membrane-bound ACh receptor (pmolligand/mg membrane protein)

Ligand Trial 1 Trial 2 Trial 3 Trial 4

F-Btx 2.63 1.47 � 0.07 1.86 � 0.24 n.t.R-132A 2.68 1.67 � 0.03 2.10 � 0.03 1.72 � 0.18R-383C 1.36 0.61 � 0.01 0.88 � 0.09 0.83 � 0.21

n.t., not tested.

Differential Surface Accessibility of �(187±199) 319

the two a subunits in the three-dimensional model,and a characterization of structural cross-talkbetween the two a(187±199) segments induced byligand binding.

Results

Stoichiometry of mAb 383C binding to AChreceptor-enriched membranes

MAb 383C titrates membrane-bound ACh recep-tor in an ELISA with a plateau value of half thatobserved for mAb 132A (Figure 1). 132A is an anti-ACh receptor mAb that competes with mAb 35(Tzartos & Lindstrom, 1980) for an epitope on theACh receptor (M. A. Agius et al., personal com-munication). 132A binds to the a subunit in aWestern blot (M. A. Agius et al., personal com-munication; Blair et al.,1988), but its binding to thereceptor is not affected by prior binding ofa-Btx. This mAb is thought to bind near the mainimmunogenic region of the ACh receptor a sub-unit.

In order to quantify the binding stoichiometry ofmAbs 383C and 132A, we ®rst puri®ed the anti-bodies from serum-free culture medium. Next, welabeled the antibodies as well as a-Btx with thehydroxysuccinimide ester of 5(and 6)-carboxytetra-methylrhodamine or 5(and 6)-carboxy¯uorescein.Finally, the labeled proteins were used in an ultra-

Figure 1. ELISA titrations with various anti-AChreceptor mAbs of ACh receptor-enriched membranesunmodi®ed and modi®ed (*) with ¯uorescein cadaverineattached to ACh receptor COOH groups. The additionof the dye has minimal affect on the binding of mostantibodies. Left panel: 132A (circles) is a competitiveinhibitor of mAb 35 binding; 383C (squares) does nottitrate a-Btx-treated, ACh receptor-enriched membranes;and 147G (diamonds) is an anti-¯uorescein mAb. Notethat the values of the plateaus of the 383C titrations areone-half the values of the plateaus of the 132A titrations.Right panel: titrations as before with 387D (circles),247G (triangles) and 371A (squares). The ®rst two mAbsbinding to the ACh receptor is affected by prior bindingof a-Btx, whereas that of 371A is not. Each of thesemAbs appears to titrate ACh receptor-enriched mem-branes with the same stoichiometry as 383C.

centrifugation assay to determine the maximumbinding to membrane-bound ACh receptor: theresults are summarized in Table 1. In each exper-iment, the maximal 383C stoichiometry measuredas picomoles of mAb per microgram of membraneprotein was one-half the value obtained for mAb132A or a-Btx. Using Fabs of 383C and 132A in anELISA titration of membrane-bound ACh receptorresulted in a plateau value for the 383C Fabtitration of one-half that of the 132A Fab titration(data not shown). Hence, the number of 383Caccessible epitopes on membrane-bound AChreceptor is one half the number of 132A epitopesor toxin sites.

Stoichiometry of 383C binding to TritonX-100-solubilized ACh receptor

Membrane-bound ACh receptor was labeledwith ¯uorescein cadaverine subsequent to acti-vation of carboxyl groups with a water-soluble car-bodiimide. The incorporation of dye was 3.6 pmolof dye per microgram of membrane protein. Thelabeled ACh receptor (F-ACh receptor) binds a-Btxand a panel of anti-ACh receptor mAbs with thesame stoichiometry and af®nity as unlabeled AChreceptor (Figure 1). Gels of the labeled receptorindicate roughly equivalent incorporation of ¯uor-escein into each of the ACh receptor subunits (datanot shown). To study the stoichiometry of 383Cbinding to Triton solubilized ACh receptor, theF-ACh receptor was extracted with 1% (v/v) TritonX-100, complexed with a threefold excess of rhoda-mine-383C (R-383C;1.0 dye/mAb) and run on a5 ml, 5% to 20% (w/v) sucrose gradient, which wascollected in 170 ml fractions. Each fraction wasplaced in SDS/borate reading buffer, and the ¯uor-escence emission of rhodamine and ¯uoresceinrecorded for each fraction. Using a standard curvefor each dye-labeled derivative, we converted thetwo ¯uorescent signals in each fraction into pico-moles of R-383C and micrograms of membrane pro-tein (Figure 2). R-383C complexes with the F-AChreceptor sediment more rapidly in the gradient thanthe F-ACh receptor alone. This allows one to collectthe complexes saturated with R-383C. The threepeak fractions of the R-383C/F-ACh receptor com-plexes in Figure 2 have an average value of thespeci®c binding activity of 383C to the solubilizedprotein of 7.2(�0.3) pmol/mg as summarized inTable 2.

Figure 2. A 5% to 20% sucrose gradient throughwhich Triton X-100-solubilized, ¯uorescein-labeled AChreceptor, complexed with rhodamine-labeled 383C wassedimented. Fraction 1 is 20% sucrose and fraction 30 is5% sucrose. Fractions are quantitatively analyzed for ¯u-orescein-labeled membrane protein (mg) and rhodamine-labeled 383C (pmol) with standard curves for bothlabeled species. For each fraction, the emission from ¯u-orescein and rhodamine was measured and convertedto either mg of membrane protein (&) or pmol of R-383C (*) in the fraction. The position of uncomplexedF-ACh receptor run on a separate 5% to 20% gradient isindicated by the arrow, and uncomplexed R-383C(pmol/5) also run on a separate gradient is presented(~) indicating no aggregate that migrates to the pos-ition of the ACh receptor/383C complexes.

Figure 3. ELISA titrations of ACh receptor-enrichedmembranes with puri®ed mAb 383C in the presenceand absence of agonist or antagonist. Both 10ÿ4 Mcarbamylcholine and 10ÿ7 M a-Btx inhibit the binding ofmAb 383C.

320 Differential Surface Accessibility of �(187±199)

To compare the 383C speci®c binding activity ofthe Triton X-100 solubilized receptor to that of themembrane-bound receptor, we prepared com-plexes of R-383C with membrane-bound F-AChreceptor and separated the complexes fromunbound R-383C by ultracentrifugation. The mem-brane pellets were extracted into 1% Triton X-100and diluted into SDS/borate reading buffer. Theratio of the rhodamine emission to ¯uoresceinemission, Fr/Ff, was determined and compared tothat observed for the complexes in the sucrose gra-dient. The Fr/Ff ratio of 0.0459 for receptor-enriched-membranes/383C complexes is one-halfthe value of Fr/Ff of 0.0941 for the Triton-solubil-ized-receptor/383C complexes from the sucrosegradient. This indicates that there are one-half asmany 383C epitopes per membrane-bound recep-tor as per Triton X-100-solubilized receptor.

Table 2. Stoichiometry of 383C binding to TritonX-100-solubilized ACh receptor

Fraction383C

(pmoles)ACh

receptor (mg)

383C pmolper mg ACh

receptorAverage

(pmol/mg)

7 0.261 0.038 6.98 0.409 0.055 7.4 7.2 � 0.39 0.296 0.040 7.4

383C titration of carbamylcholine oraaa-Btx-treated membrane-bound ACh receptor

Membrane-bound ACh receptor adsorbed tomicrotiter plates, treated with 10ÿ4 M carbamyl-choline or with 10ÿ7 M a-Btx, and titrated with383C show no binding activity of 383C to theseagonist/a-neurotoxin-treated membranes com-pared to untreated membranes (Figure 3).

Dissociation kinetics of 383C/aaa(183±199) and383C/ACh receptor complexes

We previously mapped the 383C epitope(Fairclough et al., 1998a) to a(187-199). To addressthe question of how much of the 383C epitope isrepresented by the peptide, we studied the kineticsof association and dissociation of the 383C/a(183-199) complex via surface plasmon resonance (SPR)using the BIACORE1 system, and compared theresults to the kinetics of association and dis-sociation of 383C/ACh receptor complexes. Thepeptide was covalently attached to the dextranmatrix using EDC/sulfohydroxysuccinimide acti-vation of the matrix carboxylate groups that canthen react with peptide primary amines at the Nterminus and at the e-amino group of Lys185. Theassociation and dissociation of puri®ed mAb 383Cwith this peptide-modi®ed matrix is presented inthe sensorgrams in Figure 4. The kinetic constantsare ka � 2.8 � 105 Mÿ1 sÿ1 and kd � 1.3 � 10ÿ3 sÿ1.The calculated association constant is KA � 2.1 �108 Mÿ1, which translates to �G � ÿ11.4 kcal/mol.

Figure 4. BIACORE kinetic traces for the association(A) of 25, 50, 100, 200 and again 25 nM 383C (a,b,c,dand e, respectively) with the a(183±199) peptide,GWKHWVYYTCCPDTPYL, coupled to the dextranmatrix, and the corresponding dissociation (D) kinetics.The rate constants, evaluated with the BIAevaluationsoftware, are ka � 2.8 � 105 Mÿ1 sÿ1 and kd � 1.3 �10ÿ3 sÿ1.

Figure 5. Time-course of the dissociation of mem-brane-bound ACh receptor/R-383C complexes with andwithout a-Btx bound in the low af®nity dTc site. Theamount of R-383C in the complex is assessed at thetime-points indicated by airfuge centrifugation andresuspension in an SDS/borate buffer followed byrecording the ¯uorescence emission of the resuspendedpellet. The a-Btx bound across the receptor from where383C binds weakens the contacts between 383C and itsepitope: a2(187±199).

Differential Surface Accessibility of �(187±199) 321

The dissociation of the 383C/ACh receptor com-plex was studied using rhodamine-labeled 383C(R-383C) and an airfuge separation of free R-383Creleased from the R-383C/ACh receptor complexin the presence of a tenfold excess of unlabeled383C after time t (Figure 5). We found the complexlifetime t � 171 hours, which corresponds to a kd

value of 1.6 � 10ÿ6 sÿ1. The association rate of383C to ACh receptor-enriched membranes via SPRwas found to be ka� 2 � 105 Mÿ1 sÿ1 (datanot shown). The calculated association constantfor 383C/ACh receptor complexes is thenKA � 1.25 � 1011 Mÿ1, which corresponds to�G � ÿ15.2 kcal/mol. The difference between thetwo �G values is 3.8 kcal/mol and represents theadditional stabilization of the receptor/383C com-plex compared to the peptide/383C complex. Thisvalue is very close to the 4.0 kcal/mol stabilizationenergy measured for a single hydrogen bond(Bartlett & Marlowe, 1987).

383C binding to membrane-bound AChreceptor/aaa-Btx complexes

In a membrane pelleting assay, 30 nM toxin sitesof ACh receptor-enriched membranes treated with75 nM ¯uorescein-a-Btx (F-Btx) bind only one-halfthe maximal amount of F-Btx, but are unable tobind 383C (Figure 6(a)). In a similar pelletingassay, dTc at 5 mM can inhibit the binding of180 nM rhodamine-a-Btx (R-Btx) to this ®rst site,while leaving intact the Btx binding to the second

site (Figure 6(b)), the low-af®nity dTc site. In anELISA experiment, an ACh receptor/a-Btx com-plex prepared in the presence of 5 mM dTc alsobinds 50% of the maximum F-Btx (Figure 6(c)) but,unlike the complex formed in Figure 6(a), is able tobind 383C with virtually the same stoichiometry asuntreated ACh receptor (Figure 6(d)). The 383Caf®nity is decreased slightly because the titrationcurve is shifted to higher concentrations of 383C.We observe that the dissociation rate of 383C fromthese ACh receptor/a-Btx complexes is 0.0136 hÿ1,representing a complex lifetime of 74 hours com-pared to the lifetime of 383C/ACh receptor com-plexes alone, which is 171 hours (Figure 5). Thisresult is consistent with slightly weaker bindingaf®nity of 383C in the presence of a-Btx at the low-af®nity dTc site.

aaa-Btx binding to 383C/ACh receptormembrane complexes

ACh receptor-enriched membrane vesicles satu-rated with mAb 383C bind less than 15% of thetoxin bound in the absence of 383C (Figure 7). Theresidual binding of a-Btx in the presence of 383C isvery likely a-Btx non-speci®cally associated withthe membrane preparation or trapped within themembrane vesicles, since ACh receptor treatedwith unlabeled a-Btx binds 13% of the labeledtoxin bound by untreated ACh receptor (data not

Figure 6. (a) F-Btx titration (*) of ACh receptor andsubsequent binding of R-383C (&). Titration of 30 nMtoxin sites with 75 nM F-Btx results in only half thetoxin sites ®lled, but complete inhibition of the bindingof R-383C. (b) Inhibition of R-Btx binding to the AChreceptor by increasing concentration of dTc. At 5 mMdTc only the low-af®nity dTc site is able to bind R-Btx.No R-Btx binds to the high-af®nity dTc site. (c) ELISAtitration with anti-¯uorescein mAb 147G of F-Btx boundto ACh receptor-enriched membranes untreated (*) andtreated with 5 mM dTc (&) and treated with 1 mM dTc(~). In the presence of 5 mM dTc, very nearly 50% ofthe F-Btx is blocked from binding, as indicated by theanti-¯uorescein mAb 147G titration plateau for the dTc-treated membranes being one-half that of the untreated.(d) 383C titration of the ACh receptor/F-Btx complex(*) prepared as for (c) in the presence of 5 mM dTc and200 nM F-Btx to put Btx speci®cally at the low-af®nitydTc site. The 383C titration was conducted in the pre-sence of 5 mM dTc and 200 nM F-Btx. The results arepresented along with the 383C titration (&) and the132A titration (~) of the ACh receptor alone. The pla-teaus of the two 383C titrations are virtually the same,indicating titration of the same site. However, witha-Btx in the low-af®nity dTc site, the af®nity between383C and the ACh receptor appears weaker by a factorof 3.5, since the titration curve is shifted to higher con-centrations of 383C.

Figure 7. Kinetics of binding of [125I]Btx to 383C-treated and untreated membrane-bound ACh receptor.Plateaus here indicate that one equivalent of 383Cinhibits all speci®c a-Btx binding.

322 Differential Surface Accessibility of �(187±199)

shown). The binding of 383C to the ACh receptorcompletely inhibits the speci®c binding of a-Btx tothe membrane-bound ACh receptor.

Discussion

Stoichiometry of 383C binding to theACh receptor

mAb 383C binds to residues in the peptidea(187±199) of the Torpedo ACh receptor. However,383C binds to the membrane-bound Torpedo ACh

receptor with a stoichiometry one-half that of a-Btxor mAb 132A. Solubilizing the ACh receptor withTriton X-100 doubles the number of 383C epi-topes/ACh receptor, so that in Triton X-100,a(187±199) of both a subunits is able to bind 383C.This is the same stoichiometry as that determinedby Mihovilovic & Richman (1987) with TritonX-100-solubilized receptor/383C complexes on a 5to 20% sucrose gradient. And ®nally, carbamylcho-line desensitization of the ACh receptor in mem-brane vesicles renders the epitope inaccessible forbinding 383C. These dramatic alterations in bind-ing stoichiometry from 2:1 to 1:1 to 0:l as a functionof whether the ACh receptor is Triton X-100-solu-bilized, lodged in its native membrane in theclosed resting state or in the carbamylcholine-desensitized state re¯ect changes in accessibility ofeach of the two a(187±199) peptides at the surfaceof the ACh receptor when the receptor is subjectedto these different conditions.

Contacts between 383C and aaa(187±199)account for most of the 383C/ACh receptorbinding energy

Given that the mAb 383C epitope maps to thelinear stretch of a subunit residues 187 to 199, weassessed the contribution to the total bindingenergy of 383C to the ACh receptor by just thosecontacts between 383C and the peptide a(183±199). We covalently linked the peptide to thecarboxylate groups of a dextran matrix through thea-amino group of Gly183 or the e-amino group ofLys185. Measuring the association and dissociationrates of the mAb/peptide complex, we calculatedKA for the complex formation and converted this

Figure 8. A diagram of the surface-accessibility ofa(187±199) in the two a subunits of the ACh receptor.The peptide in a2 is readily recognized by mAb 383C,whereas in a1 it is occluded from binding 383C. Giventhe large contribution of the peptide to the free energyof interaction between 383C and the ACh receptor, wefeel that this differential binding re¯ects a differentialsurface accessibility of the peptide in the two a subunits.One such model is represented here. In a2, the peptideis exposed on the surface of the receptor, whereas in a1

it is more intimately in contact with the body of thereceptor. The shorter length of the shaded region on a1

portrays the peptide loop bent up or down from its pos-ition illustrated on a2.

Differential Surface Accessibility of �(187±199) 323

to a free energy, �G(peptide/383C) � ÿ 11.4 kcal/mol. Comparing this to the kinetically determinedKA and �G(ACh receptor/383C) � ÿ 15.2 kcal/mol, one sees that the contacts between the peptideand 383C account for all but 3.8 kcal/mol, whichrepresents the energy stabilization of a singlehydrogen bond. Hence, the majority of the epitopeis provided by the linear sequence, and the single383C binding site on the native membrane-boundACh receptor is most easily rationalized by a(187±199) of the other a subunit not having surfaceaccessibility. The 2:1 stoichiometry observed fordetergent-solubilized ACh receptor would indicatethat the difference between the two sites is not achemical difference such as one site havingCys192/193 reduced and alkylated and not bind-ing 383C and the other site having these disul®de-bonded and binding 383C. Both sites, in fact, canbind 383C.

Structural distinction between the twoaaa subunits

The observed difference in binding of 383C tothe two a(187-199) segments in the membrane-bound ACh receptor closed resting state re¯ects anin situ structural difference between the two pep-tide segments in the respective a subunits: in thea2 subunit described by Kubalek et al. (1987), thea(187-199) peptide is freely accessible to the bind-ing of 383C, and in the a1 subunit the peptide isoccluded from binding 383C. The picture thatemerges from this study is presented in Figure 8,and is consistent with Unwin's (1996) ®nding thatthe two a subunits have different conformations inthe region where 383C interacts with a2. Function-ally then, a2 corresponds to which a subunit? Is a2

the subunit associated with the high-af®nity orlow-af®nity dTc binding site?

Correlation of 383C reactive aaa subunit withdTc sites

In terms of the dTc binding sites, 383C/AChreceptor complexes retain both a high and a low-af®nity binding site (Mihovilovic & Richman,1987). 383C binding does not appear to knock outone or the other dTc binding site preferentially. Infact the Scatchard plot of dTc binding to 383C/receptor complexes indicates a ®lling of the twosites at lower concentrations of free dTc concen-trations than occurs for native ACh receptor. SodTc binding is not substantially different in thepresence of 383C than its absence and, as a result,no information is available from these titrationsabout which a subunit 383C recognizes. Likewise,dTc (1 mM)-treated, receptor-enriched membranestitrated with 383C exhibit the same apparent af®-nity and stoichiometry as untreated membranes(Mihovilovic & Richman, 1987). So no informationis available from dTc inhibiting 383C bindingeither. These results do argue that residues in thea(187-199) peptide likely play a less important role

in binding dTc than residues in other parts of aand/or g and d.

Experiments that bear on the identity of the asubunit recognized by 383C are those presented inFigure 6. Concentrations of F-Btx that ®ll only oneof the two toxin sites completely inhibit the bind-ing of 383C (Figure 6(a)). This site has been ident-i®ed as the ``irreversible'' toxin site by Conti-Tronconi et al. (1990) and correlated with the BAC-reactive site by Dunn et al. (1993), and thereby tothe high-af®nity dTc site (Ratnam et al., 1986).Further evidence for such a correlation comes fromthe 383C titration of a single site on the ACh recep-tor (Figure 6(d)) when the low-af®nity dTc site iscomplexed with F-Btx (Figure 6(c)). The titrationspresented in Figure 6(d) are consistent with 383Cbinding to the a subunit associated with the high-af®nity dTc site, the site not covered with F-Btx.This then correlates the functional high-af®nity dTcsite with a2 in the structural scheme proposed byKubalek et al. (1987). This is the a subunit betweenb and g. The 383C titration curve in the presence ofF-Btx at the low-af®nity dTc site (Figure 6(d)) isshifted to slightly higher concentrations of 383C,indicating a weaker af®nity between this site and383C than that obtained in the absence of toxin. Inaddition, the dissociation rate of 383C from thissite in the presence of a-Btx in the low-af®nity siteis faster than that from the 383C/ACh receptorcomplexes formed in the absence of any toxin(Figure 5). These differences re¯ect the effect ofa-Btx bound to the low-af®nity dTc site on the

324 Differential Surface Accessibility of �(187±199)

disposition of the a(187±199) residues at the high-af®nity dTc site. The a(187±199) peptide at thehigh-af®nity dTc site forms a more stable complexwith 383C in the absence of a-Btx at the low-af®-nity dTc site than in its presence.

383C-induced structural changes at the toxinsite associated with the low-affinity dTc site

An experiment that addresses the surface acces-sibility of a(187±199) at the low-af®nity dTc site ofmembrane-bound ACh receptor is that illustratedby Figure 7. When 383C binds to the a subunit atthe high-af®nity dTc site, this binding inhibitsa-Btx binding to both a subunits (Mihovilovic &Richman, 1987; and see Figure 7). The peptidea(187±199) contains several amino acid residuesimplicated in the complex formed between theACh receptor and a-Btx (Basus et al., 1993). Clearly,383C directly blocks a-Btx binding to the high-af®-nity dTc site. But how does the single equivalent of383C inhibit the binding of the second equivalentof a-Btx? 383C could bind with each Fab arm com-bining with one of the two a(187±199) peptides.This would predict that the stoichiometry of bind-ing Fab fragments of mAb 383C to the ACh recep-tor should be the same as the stoichiometry ofbinding a-Btx or Fab fragments of mAb 132A,which binds to a main immunogenic site on each asubunit (M. A. Agius et al., personal communi-cation). Upon producing Fabs of 383C and 132A,we found the relative stoichiometry of binding tothe ACh receptor to be one Fab 383C to two Fabsof 132A.

Hence, the ability of 383C to inhibit the bindingof a-Btx at the low-af®nity dTc site must derivefrom structural changes induced at this site by383C binding at the high-af®nity site. Thesechanges are manifested across the rosette at thelow-af®nity dTc site as a further decrease in thesurface accessibility of a(187-199). Despite thischange in surface accessibility of the peptide at thelow-af®nity site, the residues of the peptide at thissite can still bind carbamylcholine, since 383C/ACh receptor complexes retain the ability to bindone-half of the carbamylcholine that untreatedACh receptor does (Mihovilovic & Richman, 1987).It appears that 383C binding to a(187±199) of thehigh-af®nity dTc site removes the low-af®nity sitea(187±199) further from the surface of the AChreceptor so that neither 383C nor a-Btx can bind.But 383C binding leaves intact the structuralorganization of the low-af®nity site peptide seg-ment, since carbamylcholine still binds to this site.

Differential surface accessibility of aaa(187±199)may explain classes of aaa-Btx sites

These data suggest that a possible structuralbasis for the irreversible and reversible a-Btx sitescharacterized by Conti-Tronconi et al. (1990) is thesurface-accessibility of residues a187 to a199. Thispeptide ®gures centrally in the binding of 383C

and a-Btx, and appears to be considerably moreexposed at the high-af®nity dTc a subunit, a2, thanat the low-af®nity dTc a subunit, a1. This differen-tial exposure could easily be responsible for thetwo different classes of toxin sites characterized byConti-Tronconi et al. (1990). We are currently prob-ing the two ACh receptor sites with ¯uorescentbungarotoxin derivatives to assess the correlationbetween af®nity for toxin and dTc.

Model of structural transitions involvingaaa(187±199)

In terms of molecular mechanics, the binding ofa-Btx to the low-af®nity dTc site seems to gentlypull the a(187±199) residues at the high-af®nitysite from the surface, so that 383C does not makeas favorable contacts with this peptide as it does inthe absence of a-Btx. This leads to faster dis-sociation of 383C/ACh receptor complexes andweaker af®nity between 383C and the ACh recep-tor. Similarly, binding of 383C at the high-af®nitysite pulls the a(187±199) peptide at the low-af®nitysite, so that not even a-Btx can bind there. How-ever, the local organization of the peptide remainssuf®ciently intact to enable the smaller carbamyl-choline molecule to bind to this site. Further con-siderations of the transitions elicited bycarbamylcholine binding are discussed byFairclough et al. (1998b).

Summary of the 383C interaction with the AChreceptor (see also Fairclough et al., 1998a)

In our study of the ACh receptor with mAb383C, we have mapped the 383C epitope to aminoacid residues 187 to 199 of the ACh receptor a sub-unit, and onto the three-dimensional model of theACh receptor, 6� clockwise of the a2 pentagonalvertex and 30 AÊ down from the top of the receptor.The contacts that 383C forms with the peptide con-tribute all but a single hydrogen bond of theenergy of 383C binding to the native ACh receptor.In this regard, 383C has been as well characterizedas, if not better than, any other anti-ACh receptorantibody.

The 383C epitope includes four amino acid side-chains implicated in the binding of acetylcholine:Y190, C192, C193 and Y198. Apart from enabling amapping of these functionally critical residues ontothe three-dimnesional model, mAb 383C dis-tinguishes between a(187±199) in the two a sub-units: in the closed resting state of membrane-bound ACh receptors, only a2(187-199) is accessiblefor interaction with 383C as depicted in Figure 8.The amino acid residues of a1(187±199) are likelynestled closely to the body of the receptor orembedded in it, thus rendering the peptide inac-cessible to the 383C combining site. We have used383C to detect differences in the disposition of thea(187±199) epitope in agonist and antagonist-trea-ted forms of the receptor compared to untreatedforms. The 383C-accessible a2(187±199) in the

Differential Surface Accessibility of �(187±199) 325

closed resting state converts to an inaccessible statein the presence of 100 mM carbamylcholine. Giventhe substantially weaker af®nity of the ACh recep-tor for carbamylcholine than 383C, we suspect thatcarbamylcholine is not merely acting as a competi-tive inhibitor of 383C, but that binding of carba-mylcholine converts the a2(187±199) peptide to aform much more like that of a1(187±199) inFigure 8: nestled close to or embedded in the bodyof the receptor and inaccessible to 383C. In the pre-sence of a-Btx at the low-af®nity tubocurarine site,the a2(187±199) epitope binds 383C with a slightlyweaker af®nity than in the absence of a-Btx, docu-menting communication between a(187±199) inthe two receptor a subunits. Also documentingsuch site to site communication is the observationthat binding of 383C on a2 inhibits the binding ofa-Btx at a1 as well as at a2. And ®nally, the TritonX-100-solubilized receptor displays both a(187±199) peptides so that 383C can bind, suggestingthat in Triton both a(187-199) peptides nowresemble a2(187-199) in Figure 8. We are currentlytesting these hypothesized molecular mechanics ofthe agonist binding regions of the ACh receptor.

Materials and Methods

Preparation of ACh receptor-enrichedmembrane vesicles

Alkali-stripped (Neubig & Cohen, 1979) receptor-enriched membrane vesicles were prepared as decribedby Fairclough et al. (1998a). For unstripped membranes,the second 18,000 rpm pellets of the stripped type prep-aration were resuspended in 2 ml of 26% (w/v) sucrosein buffer A directly without alkali treatment. This sus-pension was layered onto a 35 ml 28% to 40% (w/v)sucrose gradient in buffer A in an SW28 ultraclear tubeand spun at 23,000 rpm overnight at 4�C. The gradientwas fractionated into 1.50 ml fractions using a peristalticpump and fraction collector. Aliquots of the fractionswere assayed for percentage sucrose using a refract-ometer and for protein concentration using a modi®edLowry/Folin assay (Markwell et al., 1978). The peak pro-tein fractions were collected and diluted in buffer A, pel-leted in an SW28 rotor, and resuspended in buffer A atroughly 3.0 mg/ml. Following resuspension, the proteinconcentration was determined for each pool of AChreceptor, an SDS/polyacrylamide gel run, and a ¯uor-escent toxin binding assay performed as described forthe stoichiometry determinations. The membranes werealso assayed via ELISA for the ability of the mAb libraryto titrate the ACh receptor. Standard membrane prep-arations were found to bind between 1 and 2 pmol oftoxin per microgram of protein, whereas the alkali-stripped preparations bound 2 to 6 pmol/mg.

Fluorescent labeling of ACh receptor-enrichedmembranes

Alkali-stripped Torpedo californica ACh receptor-enriched membranes (300 ml, 4.3 mg/ml) were diluted to5 ml in buffer A and pelleted in an SW55Ti rotor at50,000 rpm for 0.5 hour. The pellet was resuspended in300 ml of 0.1 M borate buffer (pH 8.0) in which ¯uor-escein cadaverine (Molecular Probes) had been dissolved

to 1.5 mM with a resulting pH of 6.0. To this dye/recep-tor suspension was added 3 ml each of 100 mM 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide and 100 mMN-hydroxysulfosuccinimide dissolved in water, and themixture incubated for 16 hours at 4�C. The labeled mem-branes (F-ACh receptor) were diluted to 5.0 ml with buf-fer A and pelleted by centrifugation at 50,000 rpm for 0.5hour in an SW55Ti rotor. The pellet was subjected to ®vecycles of resuspension in 5.0 ml of buffer A, incubationfor 0.5 hour at room temperature, and SW55Ti centrifu-gation as before to remove non-covalently adsorbed/trapped dye. The ®nal pellet was resuspended in 300 mlof buffer A and stored at 4�C. The stoichiometry of dyeincorporation in pmol dye/mg protein was determinedby measuring light absorption at 497 nm withe497 � 81,000 Mÿ1 cmÿ1 and protein concentration with amodi®ed Lowry assay (Markwell et al., 1978) with BSAas a standard.

A sample of this labeled ACh receptor was subjectedto SDS/10% (w/v) polyacrylamide gel electrophoresis inthe presence and absence of b-mercaptoethanol. Illumi-nation with UV light showed that all four ACh receptorsubunits were equally labeled with the ¯uorescent dye(data not shown).

F-ACh receptor and unlabeled ACh receptor weretitrated with a panel of anti-ACh receptor mAbs, includ-ing mAb 383C in an ELISA. No signi®cant differencebetween labeled and unlabeled ACh receptor wasobserved for any of the tested antibodies shown inFigure 1. The F-ACh receptor was used in the antibodybinding experiment presented in Figure 2 and summar-ized in Table 2.

Purification of mAbs from cell culture supernatant

Hybridoma cells secreting mAbs were grown inHB101 serum-free medium (Irvine Scienti®c). The cellculture supernatant (200 ml) was centrifuged (3000 g forten minutes), ®ltered (0.45 mm pore size), and concen-trated under argon with a YM100 membrane in an Ami-con stirred cell. After buffer exchange with 10 mMpotassium phosphate buffer (pH 7.4), 2 ml of the concen-trate was loaded onto a DEAE Sepharose fast-¯ow col-umn (1.5 cm � 24 cm), CM-Sepharose, or a ProteinG-Sepharose column. Antibody was eluted with either alinear salt gradient from 0.0 M to 0.25 M NaCl in a totalvolume of 250 ml, a series of buffer concentration jumps,or a pH drop, respectively. In the A280 elution pro®lefrom the DEAE column with the salt gradient, antibody383C eluted as a small peak between 90 and120 mMNaCl, just before a large BSA peak (from the cell culturemedium). Fractions (5 ml) that contained pure antibodyas judged by gel electrophoresis were pooled, concen-trated in a 30 K Macrosep cell (5000 rpm in an SS34rotor), and stored at 4 �C with 0.025% (w/v) NaN3. Pro-tein concentration was determined by a modi®edLowry/Folin assay (Markwell et al., 1978) with an IgGstandard.

Fluorescent labeling of mAbs

Puri®ed mAb was ¯uorescently labeled on primaryamines with 5 (and 6)-carboxytetramethylrhodaminesuccinimidyl ester (Molecular Probes) to give rhodaminelabeled mAb (R-mAb). Then 150 ml of a 90 mM dye sol-ution in 0.1 M borate buffer (pH 8.0) or 25 ml of a 25 mMdye stock in DMF was added to 330 ml of a 20 mM anti-body solution in 10 mM phosphate buffer (pH 7.4) with

326 Differential Surface Accessibility of �(187±199)

0.025% (w/v) NaN3 and the mixture gently agitated(nutated) for one hour at room temperature. The reactionwas stopped by the addition of 50 ml of freshly prepared1.5 M hydroxylamine (pH 8.0). After 0.5 hour at roomtemperature, the dye-labeled antibody was separatedfrom free dye by gel-®ltration on a Bio-Gel P-100 column(1 cm � 19 cm) using 10 mM potassium phosphate(pH 7.4) buffer with 0.025% sodium azide to elute theantibody. The peak fractions containing antibody (voidvolume) were pooled and stored at 4�C. The stoichi-ometry of dye incorporation (pmol dye/pmol protein)was determined by measuring A554 and usinge � 80,000 Mÿ1 cmÿ1 along with a modi®ed Lowry assay(Markwell et al., 1978) with an IgG standard to measurethe mAb concentration. R-mAbs with 1 to 10 dye mol-ecules/mAb were used in the stoichiometry experimentsof Tables 1 and 2, and Figure 2.

Fluorescent labeling of aaa-Btx

a-Btx was labeled with 5-(and-6)-carboxy¯uoresceinand 5-(and-6)-carboxytetramethylrhodamine succinimi-dyl esters (Molecular Probes) to give F-Btx and R-Btx,respectively. A sample (5 mg) of a-Btx was dissolved in0.5 ml of 0.1 M borate (pH 8.0) buffer; 25 ml of 25 mMdye stock solution in DMF was added to the a-Btx sol-ution and nutated for one hour at room temperature.The reaction was stopped by the addition of 200 ml offreshly prepared 0.6 M hydroxylamine (pH 8.0). Thedye-labeled a-Btx was separated from free dye by gel-®l-tration on a Bio-Gel P-10 or P-2 column eluted with10 mM phospate buffer (pH 7.0), and the labeled toxincollected and characterized. Several batches of F-Btx andR-Btx were prepared and the pmol dye/pmol proteinwas determined by UV/VIS absorption measurements in1% (w/v) SDS borate (pH 9.3) buffer using e546(rho-damine) � 80,000 Mÿ1 cmÿ1, and e492(¯uorescein) �68,000 Mÿ1 cmÿ1 and the modi®ed Lowry assay(Markwell et al., 1978) with a-Btx as a standard. TheF-Btx derivative stock was 3.62 mg/ml with 0.88dye/toxin (or 1.54 mg/ml with 1.02 dye/toxin), and theR-Btx stock was 2.5 mg/ml with 1.0 dye/toxin.

ELISA-based mAb titrations of AChreceptor membranes

ELISA microtiter plates were coated with 100 ml of10 nM of a-Btx-binding sites of alkali-stripped mem-brane-bound ACh receptor in phosphate-buffered saline(PBS) overnight at 4�C and non-speci®c protein bindingin the wells was blocked with PBS/1% BSA for one hourat 23�C. In ligand competition experiments, 100 mM car-bamylcholine, or 100 nM a-Btx in PBS (pH 7.4) were pre-incubated with ACh receptor for one hour at 23�C andwere maintained at the stated concentrations duringmAb titrations. Mab serial dilutions in PBS were pre-pared on separate dilution plates and 100 ml of eachdilution was transferred to the ELISA plate and incu-bated for one hour at 23�C. Plates were washed threetimes with PBS and wells were incubated for one hourwith goat anti-rat IgG, heavy and light chain-speci®cantibody conjugated to horseradish peroxidase (Cappel,Organon Technika). To determine the amount of boundperoxidase, the ELISA plates were washed 12 times inPBS and 200 ml of 1 mg/ml o-phenylene-diamine dihy-drochloride (Sigma) in 0.1 M citrate buffer (pH 4.5) with0.03% H2O2 was added to each well and the absorbancewas read at 450 nm on a umax kinetic microplate reader

(Molecular Devices). When the absorbance of a controlwell containing excess mAb 132A as the primary anti-body reached a value between 1.0 and 1.5, the entireplate was read and the data for each mAb concentrationand condition plotted and stored. The smooth curvesthrough the points of the titration were determined withDelta Graph and the user de®ned formula:

Y � �AÿD�=�1� �X=C�B� �D

where A and D are the minimum and maximum plateauvalues, respectively, of the titration, C is the in¯ectionpoint of the titration and B is the Hill coef®cient of thetitration with a value near 1.0. Y is the A450 and X is theadded concentration or relative concentration of themAb. Since no quantitative conclusions are drawn fromthe position of the in¯ection point in the titrations, theadded concentrations were used for simplicity ratherthan the free mAb concentrations. Figures 1, 3, 6(c), and6(d) derive from ELISA titrations.

mAb binding stoichiometry to membrane-boundACh receptor

Three hundred and ®fty pmol of R-mAb (four or ®vedye molecules/383C; ®ve or 11 dye molecules/132A)was incubated for two hours at 4�C with 35 pmol ofmembrane-bound ACh receptor toxin sites in 100 ml PBS(pH 7.5) supplemented with 5% (w/v) BSA. Receptor/mAb complexes were isolated by airfuge centrifugationat 20 psi for 15 minutes. The supernatant was aspiratedwith a ®ne needle, and the pellet was washed twice with200 ml of cold PBS without disturbing the pellet. Pelletswere resuspended in 100 ml of 10 mM carbonate buffer(pH 8) with 1% (w/v) SDS by vortexing in a sonicatingwaterbath at 23�C. The concentration of mAb was deter-mined from the rhodamine absorbance at 554 nm or ¯u-orescence at 576 nm excited with 544 nm light using astandard curve prepared with R-mAb of different con-centrations. In trials 1 and 3 of Table 1, alkali-strippedmembranes were used, and in trials 2 and 4 non-alkali-stripped membranes were used. The number of toxinsites recovered in the pellets was determined either by[125I]a-Btx binding or by F-Btx via a centrifugation assayin parallel samples of membrane-bound receptor orreceptor/132A complexes.

383C binding stoichiometry to TritonX-100-solubilized ACh receptor

F-ACh receptor (1.2 mg) was solubilized in 200 ml buf-fer A containing 1% Triton X-100, reduced form, for onehour at room temperature. A threefold excess of R-383Cover toxin sites was added and incubated for two hoursat room temperature. The free antibody was then separ-ated from the mAb-ACh receptor complex on a 5 mlsucrose gradient prepared from 5% and 20% sucrosestock solutions in buffer A. Solubilized ACh receptor/383C complexes were airfuged for 15 minutes at 20 psito pellet the non-extracted receptor. Extracted complexes(200 ml) were layered on top of a 5 ml 5% to 20% sucrosegradient in buffer A with 1% Triton X-100, reducedform. The gradient was centrifuged in an SW 55Ti rotorat 50,000 rpm for four hours. The gradient was fractio-nated into 0.17 ml portions with a peristaltic pump anda Gilson fraction collector; the volume of each fractionwas adjusted to 2 ml with 1% SDS, 0.1 M borate (pH 9.3),and the ¯uorescence emission of rhodamine (575 nm)excited at 550 nm and of ¯uorescein (516 nm) excited at

Differential Surface Accessibility of �(187±199) 327

494 nm recorded. The top 2 ml of the gradient containedfree antibody and the bottom half mAb-ACh receptorcomplexes (see Figure 2). The amount of R-383C andF-ACh receptor in the 383C/ACh receptor complex wascalculated from standard curves, and the binding stoichi-ometry of 383C to solubilized ACh receptor determinedto be 7.2 pmol/mg.

In a parallel experiment, 200 ml of F-ACh receptor-enriched membranes (1.2 mg of protein) was combinedwith a threefold excess of R-383C and incubated for twohours at room temperature. ACh receptor/R383C com-plexes were pelleted in an airfuge for 15 minutes at20 psi and the supernatant removed. The pelleted mem-branes with R-383C bound were detergent-extracted fortwo hours at room temperature with 200 ml of 1% TritonX-100 in buffer A. The volume was adjusted to 2 ml withSDS/borate (pH 9.3). The ¯uorescence emission of bothrhodamine and ¯uorescein were recorded. The ratio of¯uorescence emission of rhodamine to ¯uorescein in thissolubilized membrane pellet was half that observed forsolubilized receptor from the sucrose gradient.

Synthesis and purification of the aaa (183±199) peptide

The 17 residue peptide (GWKHWVYYTCCPDTPYL)corresponding to the sequence a(183-199) of T. californicawas synthesized (UC Davis, peptide synthesis facility)using solid-phase peptide synthesis strategy. p-Hydroxy-methyl phenoxymethyl polystyrene resin (HMP-resin/Wong resin) was used (0.25 mmol scale) and the ®rstamino acid residue (Fmoc-leucine) coupled using dicy-clohexylcarbodiimide and the resin capped with benzoicanhydride. The synthesis of the peptide was accom-plished by repetitive cycling of Fmoc-deprotection,washing and coupling of the next amino acid (Fmoc-pro-tected, HBTU PF6

ÿ active ester). The N terminus wasdeblocked using 20% (v/v) piperidine in DMF for 30minutes at room temperature followed by a DMF washand a MeOH wash. Side-chain protecting groups wereremoved by treating the resin with a mixture of 0.75 g ofphenol, 0.25 g of 1,2-ethanedithiol, 0.5 ml of thioanisoleand 0.5 ml of water in 10 ml of tri¯uoroacetic acid forone hour at 10�C followed by one hour at room tempera-ture. This procedure also cleaves the peptide from theresin. The peptide was separated from the resin by ®l-tration through a sintered glass funnel with 10 to 20 mmpores. The resin was washed with 10 ml of dichloro-methane and the wash combined with the TFA ®ltrate.The combined ®ltrate was concentrated to 5 ml usingrotary evaporation. The peptide was precipitated with50 ml of ice-cold diethylether. The peptide was collectedby ®ltering the ether/TFA mix through a nucleopore PCmembrane (2mm pore size) and dried in a desiccator. Thepeptide was puri®ed by reversed phase high-perform-ance liquid chromatography (HP 1090) on a microsorbC4 column (4.6 mm � 250 mm, 5mm, 300 AÊ ) eluting witha linear 30 ml gradient of 0% to 60% acetonitrile in waterand each component containing 0.1% TFA. The majorpeak was collected and the identity (2132 Da) and purityof the peptide established by electrospray mass spec-trometry (mass spectral facility, UCLA).

Association and dissociation kinetics of the mAb383C/aaa(183±199) complex

The kinetics of association and dissociation betweenmAb 383C and its epitope peptide a(183±199) were stu-died using biospeci®c interaction analysis (BIA) on a

BIACORE system (see Figure 4). BIA is a biosensor tech-nology for monitoring intereactions between two ormore molecules such as proteins and peptides in realtime without the use of labels. The detection principlerelies on the optical phenomenon of surface plasmon res-onance (SPR), which detects changes in the refractiveindex of the solution close to the surface of the sensorchip. This is, in turn, directly related to the concentrationof solute in the surface layer.

The sensor chip surface consists of three layers: glass,a thin gold ®lm, and a layer of carboxymethylated dex-tran hydrogel. The hydrogel provides a hydrophylicenvironment suitable for studies of biospeci®c inter-actions and is covalently attached to the gold ®lmthrough a linker layer. It also provides a chemical handlefor covalent immobilization of biomolecules.

To perform a BIA analysis, we covalently immobilizedthe puri®ed peptide on the dextran matrix of the sensorchip. We then injected the antibody over the surface in acontrolled ¯ow. Any change in the surface protein con-centration resulting from interaction is detected as anSPR signal, expressed in resonance units (RU).

Speci®cally, 35 ml of an 11 mg/ml solution of puri®edpeptide a(183±199) in 10 mM sodium acetate buffer(pH 4.3) was immobilized on the surface of the sensorchip through the primary amine groups of the peptide,which react with EDC/SNHS-activated carboxyl groupsin the dextran hydrogel. The running buffer for thisexperiment was 10 mM Hepes-buffered saline (pH 7.4)containing 0.05% (v/v) Nonidet P-40. mAb 383C (35 ml)at concentrations of 25, 50, 100 and 200 nM in 0.01 Mpotassium phosphate buffer (pH 7.4), 0.025% azide wasinjected onto this peptide-decorated surface, and the rateof association followed (increase in RU, Figure 4, regionA), followed by a 225 second wash with running bufferto monitor dissociation of the 383C/peptide complex(decrease in RU, Figure 4, region D). The surface of thesensor chip between mAb injections was regeneratedwith 10 mM HCl. At the highest concentration of theantibody (200 nM), the dissociation was followed for alonger period of time than illustrated in the Figure. Fromthese measurements, the association rate constant (ka)and dissociation rate constant (kd) were calculated usingBIA evaluation 2.1 software (Pharmacia). An af®nity con-stant, KA, expressed as the ratio ka/kd, was calculatedfrom these values and then converted to�G � ÿ2.30RTlog KA.

Dissociation kinetics of the membrane-bound AChreceptor/R-383C complex

R-383C (800 pmol) and 160 pmoles of a-Btx-bindingsites of alkali-stripped membrane-bound ACh receptorwere incubated in 0.8 ml of 5% BSA in Torpedo Ringer'ssolution (250 mM NaCl, 5 mM KCl, 2 mM MgCl2, 4 mMCaCl2, 5 mM Tris-HCl, pH 7.4) for one hour at 23�C. ThemAb/ACh receptor complexes were collected by ultra-centrifugation in an SW55 rotor at 23,000 rpm for 30minutes. The pellet was resuspended in 1.6 ml of Ring-er's solution containing 5% BSA, 0.05% NaN3, 1 mMunlabeled mAb 383C (tenfold excess over 383C sites).The sample was placed in a 2.5 ml cryotube (Nunc)coated with 5% BSA/Ringer's solution to block non-speci®c protein binding and rocked on a nutator at 23�C.At the speci®ed times, 100 ml aliquots were removed andspun at 20 psi in ultraclear tubes of a Beckman airfugefor 15 minutes. The amount of rhodamine remaining inthe complexes was determined by resuspending the pel-

328 Differential Surface Accessibility of �(187±199)

let in 2 ml of 10 mM carbonate buffer (pH 8) containing1% SDS and measuring the ¯uorescence emission at575 nm of the sample excited at 544 nm in a SLM 4800Cspectro¯uorometer (see Figure 5).

Association kinetics of 383C binding to membrane-bound ACh receptor

The association rate constant (ka) between mAb 383Cand membrane bound ACh receptor was determinedusing SPR on the BIACORE. Alkali-stripped ACh recep-tor-enriched membranes at 100 mg/ml were dispersed bya ten second sonication in 0.01 M sodium acetate buffer(pH 4.3), and covalently coupled to the sensorchip sur-face as described above. Running buffer was HBS(pH 7.4, 0.05% Nonidet P-40). mAb 383C at 60 nM inrunning buffer was injected onto this sensorchip surfaceand the association monitored. From these data anassociation rate constant (ka) was calculated (BIAevalua-tion 2.1 software).

F-Btx titration of ACh receptor and inhibition of383C binding: a pelleting assay

Six ultraclear airfuge tubes were washed with PBS/1% BSA; 100 ml of alkali-stripped ACh receptor-enrichedmembranes at 1 mg protein/100 ml of PBS/1% BSA wereadded to each tube and titrated with 0, 25, 50, 100, 200and 300 nM F-Btx, respectively. The toxin/membranemixtures were incubated for one hour at 23�C. Thesecomplexes were then incubated with 2 � 10ÿ7 M R-383Cfor an additional hour, and the complexes pelleted in anairfuge for 15 minutes at 20 psi. The pellets were resus-pended in SDS/carbonate (pH 8) and the ¯uorescenceemission from the ¯uorescein and rhodamine deter-mined as for that from the R-383C/F-AChR complexesisolated from the sucrose gradient previously, and theresults are plotted in Figure 6(a).

Inhibition of aaa-Btx binding by d-tubocurarine

To determine the concentration of dTc that selectivelyblocks the binding of a-Btx to the high-af®nity dTc site,we titrated ACh receptor-enriched membranes with dTcand monitored the inhibition of R-Btx binding to thesedTc-treated receptors. Twelve tubes each with 18 mg ofprotein from non-alkali-stripped ACh receptor-enrichedmembranes were incubated for 30 minutes at room tem-perature with a series of increasing concentrations of dTcin 190 ml of Torpedo Ringer's solution. R-Btx (35 pmol)was then added in 10 ml to each tube, and the mixturesincubated at room temperature for 30 minutes. Themembrane complexes were separated from unboundligands by sedimentation in a Type 50.2 Ti rotor equipedwith plastic inserts that accommodate 12 airfuge-sizedtubes. These were spun at 32,000 rpm for 30 minutes.The pellets were resuspended in 2.0 ml of SDS/borate(pH 9.3). The amount of R-Btx in the complexes wasassayed by the ¯uorescence emission of the rhodaminemeasured at 575 nm excited at 550 nm. This was con-verted to pmol of R-Btx via a standard curve of the rho-damine emission prepared from known concentrationsof R-Btx in SDS/borate. The results are plotted inFigure 6(b).

Dissociation kinetics of the R-383C/AChreceptor/aaa-Btx complex

To prepare R-383C complexes with ACh receptor witha-Btx bound to the ``other'' site, we selectively blockedthe binding of a-Btx to the high-af®nity dTc with 1 mMdTc (see the inhibition curve in Figure 6(b)). Speci®cally,160 pmol of a-Btx-binding sites of alkali-stripped, mem-brane-bound ACh receptor were suspended in 0.8 ml ofTorpedo Ringer's solution containing 5% BSA , 1 mM dTcand 80 pmoles of a-Btx to block the high-af®nity dTc sitefrom a-Btx binding, but to place a-Btx on the toxin sitecorrelated with the low-af®nity dTc site. This mix wasincubated at 23�C with periodic agitation. After onehour, 800 pmol of R-383C was added and the mixturewas incubated for one additional hour, whereupon thecomplexes were collected and resuspended in a tenfoldexcess of unlabeled 383C. The kinetics of dissociation ofR-383C from the toxin/ACh receptor complexes weredetermined as for the R-383C/ACh receptor alone(Figure 5).

383C titration of F-Btx/ACh receptor complexes withF-Btx selectively at the low-affinity dTc binding site

We now transfer the results of the pelleting assay tothe ELISA experimental regimen. ELISA plates werecoated overnight with 100 ml/well of 10 nM toxin sitesof alkali-stripped, membrane-bound ACh receptor inPBS, and excess protein binding sites were blocked thefollowing day by incubation with 1% BSA in PBS con-taining 5 mM dTc. After 30 minutes the blocking sol-ution was removed and 100 ml of 200 nM F-Btx with5 mM dTc in 5% BSA in PBS was added and incubatedfor an additional hour. The amount of F-Btx incorpor-ated into the complexes was assayed by an ELISAtitration with mAb 147G, an anti-¯uorescein antibody(Figure 6(c)). The plateau level of this titration of thedTc-treated membranes is slightly more than half thatof the untreated membranes. And ®nally, the 383Ctitration of these F-Btx/ACh receptor complexes wasperformed in the presence of 200 nM F-Btx with 5 mMdTc in 5% BSA in PBS with an ELISA as with the147G titration and the results are plotted in Figure 6(d)along with the titrations of the unliganded receptorwith 383C and 132A.

Association kinetics of aaa-Btx binding to membrane-bound ACh receptor and receptor/383C complexes

To study the association kinetics of a-Btx binding tothe ACh receptor, 10 nM a-Btx-binding sites of alkali-stripped ACh receptor-enriched membranes were incu-bated alone or with 100 nM 383C for one hour at23�C, after which [125I]a-Btx (NEN) was added to eachtube to a ®nal concentration of 200 nM. At the speci-®ed times, 100 ml aliquots of the reaction mixtureswere removed and added to airfuge tubes containing a1000-fold molar excess of unlabeled a-Btx (Sigma).Bound [125I]a-Btx was determined by pelleting themembrane-bound ACh receptor in the airfuge at 20 psifor 15 minutes, removing the soup, and counting thepellets in a Beckman Gamma 2000 gamma counter; theresults are plotted in Figure 7.

Differential Surface Accessibility of �(187±199) 329

Acknowledgments

We thank Michele Mandala, Joanne Baker, DavidSanden, Nancy Barnett and Gavin Ow for excellent tech-nical assistance. We gratefully acknowledge the massspectral analysis of the peptide performed by SubhaAduru of UCLA. The ¯uorescence measurements weremade on a SLM 4800C spectro¯uorometer that was pur-chased through the University of Chicago, Departmentof Neurology with funds from the Lucille Markey Foun-dation supplemented by a Block Fund of the Universityof Chicago. The T. californica was supplied by Paci®c Bio-marine Labs, Venice, CA. The work was supported bygrants from the National Institutes of Health (NS 15462to D.P.R.) and (NS 24304 to R.H.F. and D.P.R.). G.M.T.was supported on a neuroimmunology training grant atthe University of Chicago (T32NS 07113). E.G. was sup-ported, in part, by a Kermit G. Osserman fellowshipfrom the Myasthenia Gravis Foundation, and a grantfrom the Chicago chapter of the Myasthenia Foundation.M.L. was the recipient of a Henry R. Viets fellowshipfrom the Myasthenia Foundation.

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Edited by B. Holland

(Received 2 December 1997; received in revised form 4 June 1998; accepted 11 June 1998)