Crystal structure of the extracellular domain of nAChRa1 bound to a-bungarotoxin at 1.94 A resolution

Cosma D Dellisanti1–3, Yun Yao4, James C Stroud5, Zuo-Zhong Wang4 & Lin Chen1–3

We determined the crystal structure of the extracellular domain of the mouse nicotinic acetylcholine receptor (nAChR) a1 subunit

bound to a-bungarotoxin at 1.94 A resolution. This structure is the first atomic-resolution view of a nAChR subunit extracellular

domain, revealing receptor-specific features such as the main immunogenic region (MIR), the signature Cys-loop and the

N-linked carbohydrate chain. The toxin binds to the receptor through extensive protein-protein and protein-sugar interactions.

To our surprise, the structure showed a well-ordered water molecule and two hydrophilic residues deep in the core of the a1

subunit. The two hydrophilic core residues are highly conserved in nAChRs, but correspond to hydrophobic residues in the

nonchannel homolog acetylcholine-binding proteins. We carried out site-directed mutagenesis and electrophysiology analyses

to assess the functional role of the glycosylation and the hydrophilic core residues. Our structural and functional studies show

essential features of the nAChR and provide new insights into the gating mechanism.

nAChRs mediate fast chemical transmission of electrical signals in thenervous system. They belong to the Cys-loop super family of penta-meric ligand-gated ion channels (LGICs), which also include serotonin(5-HT3), g-aminobutyric acid (GABAA and GABAC) and glycinereceptors1–3. These receptors function in the central and peripheralnervous system, and are important pharmaceutical targets for manyhuman diseases, such as myasthenia gravis, epilepsy, schizophrenia,depression and substance addiction2.

nAChRs can have different subunit compositions that give rise totheir distinct functions2. The muscle-derived nAChR is a heteropenta-mer composed of five homologous subunits, a, g, a, d and b. Eachsubunit consists of a large extracellular domain (ECD), four trans-membrane helices and a small intracellular region. The ECD ismodified through asparagine-linked glycosylations that areimportant for receptor folding, assembly and trafficking4. The ECDalso serves as the major binding site for agonists and antagonists likea-bungarotoxin (a-Btx) and other snake toxins, which are potentinhibitors of nAChRs5.

The atomic structure of nAChRs has been the focus of structuralstudies by numerous laboratories for more than a decade. In thisrespect, two major breakthroughs have been achieved so far. First,electron microscopic analyses of nAChRs from the Torpedo marmorataelectric ray organ have produced a picture of the intact channel6,7,revealing the overall organization of the pentameric assembly, thearchitecture of the ECD and the pore region of each subunit. Althoughthe electron microscopic model has provided important insights intopotential gating mechanisms8, the in-depth information offered bythis structure is limited by the low resolution (B4 A). The second

breakthrough was the discovery of an acetylcholine-binding protein(AChBP)9, a nonchannel homolog of the extracellular domain ofnAChRs. AChBP shares B24% sequence identity with nAChRs andhas the same pentameric assembly. Several crystal structures of AChBPshave been solved in different bound states, providing atomic-resolutiondetails of the interactions between the ECD and a variety of agonistsand antagonists10–14.

A central question regarding the function of nAChRs and relatedLGICs is how ligand binding is coupled to channel gating15. Extensivebiochemical and electrophysiology analyses have provided a wealth ofinformation about the role of specific residues in ligand binding andchannel function16. The Torpedo nAChR electron microscopic modeland the AChBP structures constituted a molecular framework forintegrating the vast amount of biochemical and functional dataand have led to a variety of models for the gating mechanism ofnAChR8,16–20. However, the exact mechanisms by which neurotrans-mitters induce the protein conformation changes required for channelgating are not well understood, partially because of the lack of directhigh-resolution structural data of nAChRs. Investigations have indi-cated a major role of a subunits in the channel gating process7,16. In themuscle nAChR, the ligand-binding site is formed at the a/g and a/dinterfaces, where the a subunit provides the principal binding surface(that is, the plus side)10. As seen in a series of AChBP/ligand com-plexes10,11,13, ligand binding causes a significant movement of theprincipal ligand-binding loop (loop C). In nAChR, such ligand-induced movement of loop C is thought to trigger a global conforma-tion change in the a-ECD and/or a quaternary twist between subunitsthat leads to channel opening7,17. However, no such structural changes

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Received 11 May; accepted 26 June; published online 22 July 2007; doi:10.1038/nn1942

1Molecular and Computation Biology, 2Department of Chemistry, 3Norris Cancer Center, University of Southern California, 1050 Childs Way, RIH201, Los Angeles, California90089-2910, USA. 4Zilkha Neurogenetic Institute, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, 1501 San Pablo St.,ZNI101, Los Angeles, California 90033, USA. 5University of California Los Angeles, Department of Energy, Institute for Genomics and Proteomics, 611 Young Dr. East,Los Angeles, California 90095-1570, USA. Correspondence should be addressed to L.C. (linchen@usc.edu) or Z.Z.W. (zzwang@usc.edu).

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 8 [ AUGUST 2007 953

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are observed in various AChBP/ligand complexes, raising the concernthat AChBP may represent an ‘imperturbable homologue’ of nAChRthat lacks the necessary structural elements to undergo large-scaleconformational changes in response to neurotransmitter binding21.

Here we solved the crystal structure of the entire N-terminalextracellular domain of the mouse nAChR a1 subunit (a211) boundto a-Btx at 1.94 A resolution (Supplementary Table 1 online). Thecrystal structure reveals atomic details for several key functionalelements of nAChR, such as the MIR, the signature Cys-loop and the

carbohydrate, which were not observed inprevious structural studies of AChBP.Our functional studies suggest that the carbo-hydrate is involved in a-Btx binding andchannel gating. A notable structural featureof a1 ECD is the presence of a hydrophilicpatch composed of two highly conservedhydrophilic residues and a water molecule inits hydrophobic interior. This unusual struc-tural property is absent in AChBPs, whichcontain hydrophobic residues at the corre-sponding positions. Our structure-guidedmutagenesis and patch-clamp recordingexperiments suggest that the hydrophilicpatch inside the beta sandwich of nAChRis indeed important for agonist-inducedchannel opening.

RESULTS

Overview of the structure

The structure of a211 consists of a ten-stranded b-sandwich and an N-terminala-helix (Fig. 1). The sheet made of strandsb1, b2, b3, b5, b6 and b8 is referred to as theinner sheet, as its lower part faces the axis ofthe channel in the fully assembled pentamer(Supplementary Fig. 1 online), whereas thesheet made of strands b4, b7, b9 and b10 isreferred to as the outer sheet. The outer sheetinteracts with an extended oligosaccharidechain that is covalently attached to Asn141.A disulfide between Cys128 on the inner sheetand Cys142 on the outer sheet links the twosheets together. The outer sheet and its asso-

ciated loops are also the binding sites for a-Btx, which consists of acentral b-sheet, three finger-like loops and a C-terminal loop. Itsstructure is nearly identical to that observed in a toxin/peptidecomplex22. Loops emanating from both ends of the b barrel of thea1 ECD have important roles in the physiological function of nAChR(Fig. 1a,b). At the top of the b barrel (membrane distal), loops b4-b5(loop A), b7-b8 (loop B) and b9-b10 (loop C) serve as the principalligand-binding elements at the a/g and a/d interfaces in the nAChRpentamer7,10,23. At the bottom of the b barrel, loops b1-b2, b6-b7 (the

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MIRa

c

b

Cys

1

mA1

mA3mA4mA2mA7mBmGmDLym

tA1

mA1

mA3mA4mA2mA7mBmGmD

tA1

mA1

mA3mA4mA2mA7mBmGmDLym

Lym

tA1

mA1

mA3mA4mA2mA7mBmGmDLym

tA1

10

60 70

120 130 140 150 160

170 180 190 200 210

80 90 100 110

H1 β1 β2

β2 β3 β4H3–102H3–101 β5 β5′

β6′β6 β7 β8

loop A

loop B

β10β9 loop C

Sugar Toxin Toxin & sugar Buried hydrophilic

E

R A

20 30 40 50

B C I

I

II

III

C

C

N

N N

MIR

CN

Figure 1 Overall structure of the mouse nAChR a1

subunit (cyan) bound to a-Btx (orange). The

carbohydrate chain is shown as a stick model and

colored in magenta. This color scheme is used

throughout the illustration, except where otherwise

stated. (a) Front view between the inner and outer

sheets. (b) Top view. (c) Sequence alignment of

representative nAChR subunits (A, alpha subunit;B, beta subunit; D, delta subunit; G, gamma

subunit; Lym, Lymnaea stagnalis AChBP; m,

mouse; tA1, Torpedo alpha subunit). Residues

binding to a-Btx are highlighted in orange,

residues contacting carbohydrate are in pink,

and residues contacting both sugar and toxin are

in green. The two buried hydrophilic residues

are shaded in gray. The three residues mutated

in a211 are indicated by red letters above the

native sequence. The glycosylation site Asn141

is shaded in beige.

954 VOLUME 10 [ NUMBER 8 [ AUGUST 2007 NATURE NEUROSCIENCE

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Cys-loop), and b8-b9 form the interface with the transmembranedomain and have been implicated in coupling ligand binding tochannel opening16.

The fact that a211 is crystallized as an isolated monomer in complexwith a-Btx raises the question of whether the lack of the pentamericassembly affects its structure, particularly in regions that are expectedto interact with the adjacent subunits. To address this question, wecompared the structure of a211 with that of AChBP and the electronmicroscopic model of the Torpedo nAChR. The b sandwich core ofa211 superimposed very well with the corresponding region in theintact Torpedo nAChR (pdb code 2BG9)7, AChBP bound to carbamyl-choline (AChBP-CCh, pdb code 1UV6)11 and AChBP bound toa-cobratoxin, a-Ctx (AChBP-Ctx, pdb code 1YI5)12, whereas theloops at both ends showed slightly different trajectories (Fig. 2a).The predicted pentamer assembly interfaces of a211 (usually referredto as minus and plus side) also resemble the corresponding interfaces inTorpedo nAChR and AChBP7,10. On the minus side, the structuralsimilarity extends from the main-chain conformation to the side-chainorientation (Fig. 2b). On the plus side, only minor differences wereobserved. The side chains of Thr150, Tyr151, Asp152 and Ala155(mutated from Val in a211), all in loop B of a211, are oriented as inthe Torpedo nAChR a subunit with slight offset. However, this offsetwas not observed in the comparison with both AChBP structuresconsidered here, whose plus-side side-chains aligned very well withthose of a211 (Fig. 2c). Comparison with other published AChBPstructures yielded similar results (data not shown).

A notable difference was observed for Tyr127, which is part of theplus-side interface. In Torpedo nAChR, a-Tyr127 protrudes towardthe adjacent subunit, where it could form a hydrogen bond to anasparagine (g-Asn38, d-Asn41)7. The tyrosine is replaced in AChBP bya serine (Ser122), which is within hydrogen bonding distance (B2.8–2.9 A) of an asparagine on the minus side of the adjacent subunit(Asn37)10. In our structure, the electron density of Tyr127 showstwo alternative conformations (Fig. 2d), one of which points awayfrom the adjacent subunit to form an intrasubunit hydrogen bond

with the side-chain of Glu129 (distance is B2.6 A), whereas theother is similar to the orientation seen in the Torpedo nAChRpentamer (indicated by a red arrow in Fig. 2d). The flexibility ofTyr127 observed in our crystal structure could be due to the lack of theadjacent subunit.

Overall, the structural comparison between a211 and TorpedonAChR and the AChBP pentamer complexes indicated that themonomeric state of a211 in our crystal structure does not significantlyaffect the folding and most of the side-chain conformations of thesubunit, which likely resembles the unaltered mouse nAChR a1extracellular domain.

Binding of a-Btx to a211

Structural studies of neurotoxin binding to nAChR have so far only beencarried out with a-Btx bound to peptides or peptide mimics fromnAChR22,24, in addition to a 4.2 A crystal structure of a-cobratoxinbound to AChBP12. Our structure is the first high-resolution complex ofa-Btx bound to the entire extracellular domain of a nAChR subunit. Asfor the structural analyses of a211 described above, the toxin/receptorinteraction observed in our crystal structure could also be affected by thelack of the adjacent subunits. Furthermore, a residue known to berelevant to agonist binding, Trp149, was mutated in a211 to arginine toachieve crystallization23. This residue is located near the toxin-bindingsite. Despite these potential perturbations, a-Btx bound to a211 with ahigh affinity (Supplementary Fig. 2 online). The dissociation constantKd (0.27 nM) was very close to those observed previously for the wild-type mouse a211 (Kd ¼ 0.2 nM) and intact Torpedo or musclenAChRs25, but was considerably lower than those for bacteria-expresseda1 extracellular domains (Kd4130 nM)26 or synthetic peptides corre-sponding to the a1 subunit sequence (Kd ¼ 10 mM)27.

The a211/a-Btx binding interface is composed of a series of inter-locked loops that bury about 1,780 A2 of solvent-accessible area(Fig. 1b). Loop C of a211 is wrapped around by fingers I, II and theC-terminal loop of a-Btx, whereas the tip of the a-Btx finger II insertsinto the ligand-binding site surrounded by loops A, B and C of a211.

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Cyan: α211Red: Torpedo AChR α-subunitYellow: AChBP-CtxGrey: AChBP-CCh

Lys107

a

c d

bHis79

Thr106

Pro121

Ile38

Ile41

Asn42

Tyr151

Tyr127

Ala155

Asp152

Thr150

Figure 2 Structural comparison between a211,

Torpedo nAChR and AChBP. (a) Superposition

of the b sandwich core of a211 with the

corresponding region in the Torpedo nAChR

a subunit (r.m.s. deviation: 2.327 A with

chain A, 2.136 A with chain D), AChBP bound to

carbamylcholine (r.m.s. deviation, 1.290 A), and

AChBP bound to a-cobratoxin (r.m.s. deviation,1.394 A). (b) Detailed comparison of the minus-

side interface showing that residues involved in

subunit-subunit interactions aligned very well.

(c,d) Detailed comparison of the top (c) and

bottom (d) parts of the plus-side interface

showing that residues involved in subunit-subunit

interactions occupy similar positions, but show

slight offset. The electron density shown in d also

indicates two alternative conformations (red

arrow) of the side chain of Tyr127. The displayed

interface residues are identical between a211

(blue) and Torpedo nAChR a subunit (pink),

except for position 79 (His and Arg, respectively)

and 155 (Ala and Lys, respectively). The

corresponding AChBP residues (both complexes

in gray) are different both in type and numbering,

and are not labeled for clarity.

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 8 [ AUGUST 2007 955

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Finger I is sandwiched by the sugar and loop C, whereas finger III doesnot contact a211.

Superposition of the a211/a-Btx complex to the Torpedo nAChR(Supplementary Fig. 1) and to the AChBP/a-Ctx complex (Supple-mentary Fig. 3 online) showed that the toxin would fit between thesubunit interfaces, where finger III would make modest clashes withportions of the b8-b9 loop and the b2 strand of the adjacent subunit.These clashes suggest that the relative orientation of a-Btx in binding tothe isolated a211 may be slightly different from its binding to the intactnAChR pentamer. Indeed, compared with the position of a-Ctx in thecrystallized complex with AChBP, a-Btx is slightly tilted (B10 degrees)anticlockwise relative to a211 (Supplementary Fig. 3). On the otherhand, the chemical features of the a211/a-Btx interaction were verysimilar to those observed in the AChBP-Ctx complex, but at a muchhigher resolution.a-Btx binds to a211 through extensive hydrogen bonds and van der

Waals contacts. Many of these interactions are between main-chainatoms, giving rise to an unusually tight binding interface (Supplemen-tary Table 2 online). At the core of the ligand-binding pocket, Phe32and Arg36 of a-Btx finger II and Tyr198, Tyr190 and Tyr93 of a211forms an elaborate cation-p interaction system (Fig. 3a)28. Theguanidinium group of Arg36 (a-Btx) occupies roughly the sameposition of the quaternary ammonium of agonists when a211 wassuperimposed on AChBP bound by carbamylcholine (Fig. 3b)11,13.Other finger II residues (Asp30, Val31, Lys38, Val39 and Val40) interactwith several a211 residues, including Val188, Ser191, Cys192 andPro194 in loop C, and Asp99 and Phe100 in Loop A. On the backsideof a211 loop C, Phe189 inserts into a hydrophobic pocket formed byfinger I, finger II and the C-terminal loop of a-Btx. Similarly, at thetoxin C terminus His68 and Pro69 of a-Btx forms a sandwich-likehydrophobic interaction with Pro194.

The a211/a-Btx interface also shows an interaction that was notobserved in previous structures of toxin bound to receptor peptides orAChBP12,22. In its complex with AChBP, a-Ctx finger I interacts withprotein residues on loop C of AChBP. In our complex, a-Btx finger I isanalogously positioned relative to a211, but its interactions with thereceptor are mostly mediated by the N-linked carbohydrate chainattached to a211. Here Trp187 on a211 loop C seems to be important,as does the AChBP Trp184 in the AChBP-Ctx complex. However,Trp187 makes little direct contact to a-Btx, but stabilizes the sugarconformation from MAN5 to MAN8 in such a way that these

mannoses interact with a-Btx through numerous polar contacts withThr6, Ala7, Thr8 and Ser9 of a-Btx (Fig. 3c and SupplementaryTable 3 online). Therefore, the sugar seems to be important in thebinding of the toxin to the muscle nAChR.

To further address the function of the carbohydrate in toxin binding,we treated cell surface–expressed muscle nAChR with endoglycosidaseH (Endo H). Deglycosylation reduced the toxin binding to nAChR bymore than two orders of magnitude (Kd ¼ 1.02 � 10�11 versus 7.3 �10�9 M for untreated and Endo H-treated, respectively; Supplemen-tary Fig. 2). Treatment with Endo H did not alter the Bmax in the toxin-binding assay, indicating that the total number of nAChRs wereunchanged on the cell surface. These findings are consistent with ourstructural observation that a-Btx binding to nAChR involves extensivecontacts to the carbohydrate.

MIR

The b2-b3 loop of a211 harbors the MIR (residues 66–76) that bindsautoimmune antibodies from patients with myasthenia gravis. TheMIR is located at an upper protruding point near the N terminus of thereceptor (Fig. 1a,b). Its conformation is held by a 310-helix andintimate interactions with the N-terminal a-helix and the b5-b6 turn(Fig. 4a). Trp67 is anchored into a hydrophobic core between Leu7 andLeu11 on the N-terminal helix and Tyr112 on the b5-b6 turn, whereasAsp71, Tyr72, Gly73 and Val75 are packed closely with His3, Glu4,Arg6, Leu7 and Lys10 on the N-terminal helix through extensivehydrogen bonds and van der Waals contacts. The MIR involves littlecrystal contact and is located away from the pentamer assemblyinterface. Thus, the MIR in the intact channel likely assumes a similarstructure as observed here. The well-defined conformation of MIR andits intimate association with neighboring protein structure areconsistent with the fact that most autoimmune antibodies inmyasthenia gravis require the native receptor to bind with highaffinity29 (Supplementary Fig. 4 online). The structural informationpresented here may facilitate the development of therapeutic treat-ments for myasthenia gravis.

The Cys-loop

nAChRs are also known as Cys-loop receptors because they contain a13-residue peptide linked by a cysteine disulfide16. The sequence of theCys-loop is highly conserved in LGICs, but differs substantially fromthe corresponding region of the nonchannel homolog AChBP (Fig. 1c),

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a b cP194 P197F189

C193

C192Y198

T148Y190

R149

Loop B

Loop C

MAN7MAN6

MAN5MAN8

W187

F189

P10

S9T8

T6

A7

CCh

Arg36

Y93

R36

F32

D30

Figure 3 Binding of a-bungarotoxin to a211. (a) Detailed view of the interactions at the aromatic cage. Here, Tyr198 of nAChR a1 and Arg36 and Phe32

of a-Btx stack face-to-face, whereas Tyr190 and Tyr93 of nAChR a1 pack face-to-edge with Arg36 and Phe32 of a-Btx, respectively. Arg36 of a-Btx also forms

hydrogen bonds with the main chain carbonyls of Thr148 and Arg149 of nAChR a1, whereas its carbonyl forms a hydrogen bond with the main chain amide of

Cys192 of nAChR a1. Tyr190 of nAChR a1 also forms a hydrogen bond with Asp30 of a-Btx. (b) The guanidinium group of a-Btx Arg36 (red) occupies roughly

the same position of the quaternary ammonium of carbamylcholine (yellow) when a211 (blue Ca trace) is superimposed on AChBP-CCh (gray Ca trace).

(c) Interactions between sugar and finger I of the toxin. The sugar moiety interacts with a series of small hydrophilic residues on a-Btx through main chain

hydrogen bonds.

956 VOLUME 10 [ NUMBER 8 [ AUGUST 2007 NATURE NEUROSCIENCE

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suggesting that it may confer channel-specific functions in LGICs19,30.The Cys-loop (residues 129–141 in a211) contains a sequence patternof Ar-Pro-Ar-Asp (Phe135-Pro136-Phe137-Asp138; Ar, aromatic resi-due) that has a high propensity to form type VI turns31. Indeed, theCys-loop in our structure assumes a type VIb turn, wherein Pro136adopts the characteristic cis-peptide conformation (Fig. 4b). Phe135,located at the tip of the Cys-loop, is important in maintaining thisunique conformation by packing face-to-face with the pyrrolidine ofPro136. Consistent with its key structural role, Phe135 has been shownto be essential for the function of nAChR by mutational studies32.Phe137 makes numerous van der Waals contacts with the main-chainatoms of Val132, Thr133, His134, Phe135 and Pro136, which furtherstabilize the intrinsic structure of the type VIb turn and the trajectory ofthe Cys-loop. As a result, the electron density of the Cys-loop is welldefined, despite making few contacts to the rest of the protein(Supplementary Fig. 5 online). A study of the 5-HT3A receptorhas suggested that cis-trans isomerization of a highly conserved prolineon the M2-M3 loop (Pro272) is important in channel gating19.Notably, Pro136 in the Cys-loop, which is close to the conserved prolineon the M2-M3 loop, adopts the relatively less common cis-conformation. However, the functional significance of cis-Pro136 isnot clear at present.

The electron microscopic model of Torpedo nAChR predicts that theCys-loop interacts with the loop between transmembrane helices M2and M3 (ref. 7). Indeed, the structural alignment of a211 with TorpedonAChR (Fig. 2a and Supplementary Fig. 1) shows that the Cys-loopin a211 would likely contact the upper end of the transmembranehelices, in close vicinity to the M2-M3 loop. The structural comparisonalso shows notable differences between the Cys-loop observed inisolated a211 and its counterpart in the electron microscopic modelof Torpedo nAChR. This observation suggests that the transmembranedomain can indeed affect the structure of the Cys-loop in the intactchannel, and that the interaction between the Cys-loop and thetransmembrane helices could modulate the gating process as hasbeen suggested previously8.

N-linked carbohydrate chain

The Cys-loop contains a consensus asparagine-glycosylation site (Asn-X-Ser/Thr) that is highly conserved in muscle-specific nAChRs and asubset of other pentameric LGICs (Fig. 1c). Glycosylation is known tobe important for receptor expression and trafficking. Several studies

have also shown that glycosylation might havea role in channel gating and desensitiza-tion4,33. However, little is known about thestructure and functional mechanism of carbo-hydrate in nAChRs. Our crystal structure ofa211 shows a long carbohydrate chain linkedto Asn141 (Fig. 5) with a very well-definedelectron density (Fig. 5a). The carbohydratemakes few crystal contacts. Its structure isstabilized largely by interactions with a211and a-Btx. The carbohydrate chain branchesupwards along the outer sheet, away from thepentameric assembly interfaces as revealed bythe superposition of a211 on the electronmicroscopic model of Torpedo nAChR (Sup-plementary Fig. 1). The oligosaccharide con-tains two N-acetylglucosamines (NAG2–3)and eight mannoses (MAN4–11) organizedin a typical high-mannose pattern (Supple-mentary Fig. 6 online). This pattern of

N-linked glycosylation core is conserved in mammalian cells34,35.The carbohydrate moiety adopts an extended conformation and

engages in extensive interactions with a211 strands b9 and b10, as wellas with a-Btx (Supplementary Tables 3 and 4 online). Notably, thesugar chain reaches near the backside of loop C, and thus may act as adirect link between the membrane interface at the bottom of theb barrel and the ligand-binding site (Supplementary Fig. 1). The sugar/receptor interface is largely planar with most interactions involving vander Waals contacts between sugar rings and the receptor surfaceresidues (Fig. 5b). NAG2, NAG3 and MAN4 pack face-to-face againstHis204 and Trp184, whereas MAN5, MAN6 and MAN7 interact withTrp187 near the backside of loop C. Numerous water-mediatedhydrogen bonds form between NAG2 and NAG3 and protein residuesGlu180, His204, Thr202 and Trp184. There were only two directhydrogen bonds between the carbohydrate and the receptor. One isfrom MAN5 to the main chain of Trp187 and the other is from NAG3to His186. These structural features suggest that the observed protein/sugar interactions could tolerate a certain degree of sequence variationon the receptor subunit, as has been observed in many other protein/carbohydrate complexes35. Thus, other nAChR subunits with theconserved glycosylation site likely have a similar protein/sugar complexstructure to the one observed here.

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R209 P136

D138

Q208

D139K179

F135

H134

T133

F137

V132

I131

N141S-S

K76Y112

V75

P69

D70N68

D71

L7 L11

Y72

a b

W67 K66 β3

β5

β9β10

β7

β6

Figure 4 Structure of the MIR and the Cys-loop. (a) Detailed interactions between the MIR (residues

66–76), the N-terminal helix and the b5-b6 turn. The top extremity of the MIR folds into a 310-helix

from Pro69 to Gly74. In addition to the interactions described in the text, Pro69 and Lys76 also make

van der Waals contacts to Tyr112. (b) Detailed interactions in the Cys-loop and between the Cys-loop and

the surrounding region. An interaction network between Asp138, Glu139, Lys179, Gln208 and Arg209

forms between the Cys-loop, b9 and b10 together.

H204 w91

w77w47

W184

H186

W187

CB

B

C

A

NAG2

NAG3

MAN9

MAN10

MAN11 MAN4 MAN8

MAN6

MAN5

MAN7

a b

Figure 5 Structure of the carbohydrate chain and its interaction with

the receptor. (a) Electron density of the sugar from simulated omit map

contoured at 3.0 s level. (b) Representative interactions between sugar,

receptor protein and ordered water molecules. W, amino acid Trp; w, water

molecule. a-Btx is omitted for clarity.

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Functional roles of glycosylation

To address the potential functions of the carbohydrate, we generatedglycosylation-defective mutants (Asn141Ala and Ser143Ala) in themuscle nAChR a1 subunit (see Methods). Both mutants failed toexpress on the cell surface (data not shown) and generated no currentin response to acetylcholine (Fig. 6), confirming the well-known role ofglycosylation in the folding and trafficking of the receptors4. To see ifthe carbohydrate has other functions after the receptor is transported tothe cell surface, we carried out deglycosylation studies (Fig. 6a,b).Patch-clamp recordings showed that macroscopic currents acquired inthe whole-cell configuration were markedly reduced following Endo Htreatment of transfected cells expressing the wild-type nAChR (Fig. 6c).Single channel recording in the cell-attached configuration revealedthat deglycosylation reduced the channel open-interval duration andincreased the closed-interval duration. As a result, there was a notablereduction in the probability of nAChR channel opening when the sugarwas removed or truncated (Fig. 6d,e). More-over, we found that deglycosylation had littleeffect on the affinity of nAChR to smallcholinergic ligands in binding assays using

3H-acetylcholine as a radioligand (data not shown). These findingssuggest that the carbohydrate may have an indispensable role incoupling agonist binding to channel gating. However, it should benoted that we cannot rule out the effect of deglycosylation on non-asubunits, as Endo H may remove sugars from all five subunits.

Residues coupling agonist binding to channel gating

A number of residues on the outer sheet, including Tyr93, Asn94,Lys145 and Asp200, have been implicated in channel function36,37. Theside-chains of these residues are well defined in the present structure(Fig. 7a). Lys145 and Asp200 engage in direct electrostatic interactionwith each other, and both form hydrogen bonds with Thr202. Notably,Lys145 is 8 A apart from the side-chain of Tyr190. These structuralfeatures are consistent with previous observations that the mutualinteraction and arrangement of these residues are associated with theclose state of the channel16,37,38. This is in agreement with the fact that

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Figure 6 The effect of N-linked glycosylation on the electrophysiological properties of nAChRs. Deglycosylation was carried out by treating the cells in tissue

culture plates with Endo H in DPBS for 20 min at 37 1C. (a) Western blotting with mAb210 showed the efficient cleavage of N-linked glycosylation from

nAChRs on COS cell surface by Endo H. Lane 1, detergent extracts of COS cells expressing the wild-type nAChRs. Lanes 2 and 3, surface nAChRs in the

cells were biotinylated and pulled-down by streptavidin-sepharose beads. (b) Staining with rhodamine-conjugated Con A showed that Endo H removed most

high-mannose oligosaccharides from the cell surface. (c) COS cells were transiently transfected with the wild-type or glycosylation-deficient nAChR a subunit

in combination with the wild-type b, d and e subunits. Whole-cell currents were elicited by superfusing the cells with 50 mM ACh. Membrane potential was

held at –70 mV. (d) Single-channel currents were recorded in the cell-attached configuration (50 mM ACh; open is down, filtered at 10 kHz for display). Thepotential of the pipette was held at +70 mV. Right, open- and closed-interval duration histograms. (e) Dose-response curves of the probability of being open

within a cluster (Popen). Each symbol is one patch.

Y127

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Figure 7 Mapping functional residues on the

structure. (a) A view normal to the outer sheet

near the ligand-binding site; residues implicated

in channel functions interact with each other and

with the carbohydrate chain. This view also shows

the entrance of the hydration cavity (red arrow)

deep in the hydrophobic core between the innerand outer sheets. (b) View from the bottom of the

b barrel at the membrane interface. Residues

from different loops that interact with each other

are highlighted.

958 VOLUME 10 [ NUMBER 8 [ AUGUST 2007 NATURE NEUROSCIENCE

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here a211 is bound by an antagonist. Thus, its overall structure maycorrespond to the closed state39. Our high-resolution structure alsoshows detailed interactions of these functionally important residueswith other receptor residues and the carbohydrate chain. Asp200 formsa hydrogen bond with His186, which in turn interacts with NAG3,MAN4, MAN5 and MAN8 through a hydrogen bond and numerousvan der Waals contacts. The aliphatic side-chain of Lys145 also packsagainst the aromatic ring of Tyr93, whereas the adjacent Asn94 formshydrogen bonds with the main-chain carbonyl and amide of Ser143and Lys145. These interactions are located on the outer sheet of thenAChRa1 subunit and away from the pentamer interface (Fig. 7a andSupplementary Fig. 1). This network of interactions may participate inthe transmission of agonist-initiated gating signal.

At the membrane interface, the three membrane-facing loops (theb1-b2 loop, the Cys-loop and the b8-b9 loop) and the premembranelinker interact with each other extensively (Fig. 7b). A number ofresidues, such as Arg209, Asp138 and Asp45, have been shown to beimportant for channel function by site-direct mutagenesis20,30,40. Inour crystal structure, Arg209 is surrounded by Asp138, Glu175 andGlu45, whereas its aliphatic side-chain packs against Trp176, which inturn makes van der Waals contacts to Leu40 and Val43. Asp138,

Glu139, Lys179 and Gln208 interact with each other through hydrogenbonding and electrostatic interaction (Fig. 4b), whereas Gln48, Tyr127,Cys128, Glu129 and Ile130 form another interaction cluster (Fig. 7b).Although the exact structure of the membrane-facing loops may beaffected by the membrane domain in the intact channel, the chemicalcomplementarity described above could still be important in theinteraction between the ECD and the transmembrane domain duringligand-induced gating.

A hydration pocket inside the b sandwich core of nAChR

At 1.94 A resolution, many ordered water molecules on the proteinsurface are visible. To our surprise, we also found a well-ordered watermolecule inside the a1 ECD. This water molecule is bonded bytwo buried hydrophilic residues, Thr52 and Ser126, respectively,and forms a hydration pocket inside the b sandwich core of thenAChR subunit (Fig. 8a). This hydration pocket is connected to theoutside through another ordered water molecule to reach the surfaceresidue Asn94 on loop A (Figs. 7a and 8a). Both hydrophilic residues(Thr52 and Ser126) are highly conserved in nAChR, whereas thenonchannel homolog AChBP has bulky hydrophobic residues (Val/Leu and Phe) at these positions (Fig. 1c). Such an unusual structural

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–2 –1–3–4Log [duration (s)]

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Figure 8 Electrophysiological properties of mutant nAChRs on the surface of transiently transfected COS cells. (a) A view from the top of the b barrel showing

the pathway of the hydration cavity along a train of hydrogen bonds between Thr52, w69, Ser126, w89 and Asn94. In this figure a-Btx is omitted for clarity.

(b) Whole-cell currents were elicited by superfusing the cells with 50 mM ACh. The membrane potential was held at –70 mV. (c) Single-channel currents were

recorded in the cell-attached configuration (50 mM ACh; open is down, filtered at 10 kHz for display). The potential of the pipette was held at +70 mV. Right,

open- and closed-interval duration histograms. (d) Dose-response curves of the probability of being open within a cluster (Popen). Each symbol is one patch.

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 8 [ AUGUST 2007 959

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feature that is unique to nAChR could be a key element required for thereceptor function as a ligand-gated ion channel.

To address this question, we converted both Thr52 and Ser126 in thenAChR a1 subunit to their hydrophobic counterparts in AChBP andanalyzed the mutational effect in intact nAChR by patch-clampexperiments. Because AChBP and the nAChR a1 ECD share a similarfold, these hydrophobic reverting mutations are not expected to affectthe folding of the a subunit and may even further stabilize it. Indeed,immunofluorescent staining with mAb35 demonstrated that nAChRscontaining any of the three a1 mutants, Thr52Val, Ser126Phe andThr52Val/Ser126Phe, were stably expressed on the cell surface inamounts comparable to the wild-type receptors (SupplementaryFig. 7 online). However, whole-cell and single-channel recordingsshowed that the mutations substantially reduced ionic current flowingthrough the cell membrane, probably as a result of the impaired abilityof nAChRs to open in response to acetylcholine. Moreover, themutational effect correlated very well with the level of hydrophobicsubstitution (Fig. 8b–d). Although both single mutants, Thr52Val andSer126Phe, showed modest, but substantial, effect, the double mutant(Thr52Val/Ser126) showed much a stronger effect. These observationsstrongly suggest that the hydrophilic interior of nAChR is importantfor channel function, probably by conferring the receptor-specificstructural flexibility required for the allosteric channel gating. Theb sandwich core residues have been previously analyzed by extensivemutational studies in light of the agonist-induced intersheet movementof the nAChR a subunit observed in electron microscopic analyses8.Most of these mutational results are difficult to interpret becauseprotein core residues are intimately associated with the folding stability.However, the observation that the Ser126Ala and the Ser126Valmutations showed little effect on gating equilibrium is consistentwith our results here, as the smaller side-chain of Ala and Val wouldprobably still leave the hydrophilic pocket unfilled32.

DISCUSSION

Structural analysis of nAChR at a high resolution has been a long-standing challenge. The structure of the extracellular domain of themouse a1 subunit in complex with a-Btx presented here provides thefirst high-resolution view of a substantial component of nAChRs.A number of receptor-specific elements, such as the MIR, the N-linkedglycosylation moiety and the signature Cys-loop, are shown atthe atomic level for the first time. Direct comparison of the a211structure with the high-resolution structures of AChBP in differentcomplex forms and with the low-resolution electron microscopicmodel of the Torpedo nAChR shows that the lack of pentamericassembly did not alter appreciably the overall fold and fine structureof a211. Thus, the a211 structure observed here can be consideredto reliably represent the structure of the nAChR subunit extra-cellular domain.

In contrast to protein residues, the function of carbohydrate onthe nAChR has attracted little attention so far4,41. An asparagine-linked oligosaccharide defined here interacts extensively withthe receptor and toxin to form an integral part of the extracellulardomain. The oligosaccharide chain emanates from the membrane-facing Cys-loop and reaches over to interact with the membrane-distalligand-binding loop, raising an intriguing possibility that the sugarchain may participate in coupling ligand binding to channel gating(Supplementary Fig. 1). Our studies suggest that glycosylationmay have multiple roles in nAChR function. Consistent with previousreports4,41, our mutational analyses and deglycosylation studiessuggest that the carbohydrate is important for the surfaceexpression of the receptor and for the high-affinity binding by

neurotoxin. Notably, deglycosylation did not change the bindingaffinity of the receptor to small cholinergic agonists, but substantiallyimpaired the ability of nAChR channels to open in responseto the agonists. Previous studies have shown that deglycosylation haslittle effect on the stability of folded nAChRs42. Thus, deglyco-sylation may have specific effects on the gating mechanism ofnAChRs. However, with the available data, this implication of glyco-sylation in gating cannot explain why the glycosylation site onthe Cys-loop is conserved only in muscle-specific nAChR, whereasother members of the nAChR family (for example, a7) have glycosyla-tion sites at different locations (Fig. 1c). A more thorough under-standing of the general role of glycosylation in channel functionrequires future studies.

A puzzling aspect of structural studies of AChBP is the lack ofsubstantial structural changes that may explain the allosteric mechan-ism by which ligand binding induces channel opening in nAChR. Themain physiological function of AChBP is to bind acetylcholine, but notto act as an ion channel9. It is most likely that the structure of AChBPhas not been optimized through evolution for channel function. Wereasoned that structural features that are highly conserved in nAChRs,but absent in AChBPs, might be involved in ligand-dependent gating ofthe ion channel. Detailed examination of the crystal structure shows ahydration cavity near the Cys-loop disulfide that penetrates deeply intothe hydrophobic core of the nAChRa subunit (Fig. 7a and 8a). Thisunusual structural feature suggests that nAChRs have a nonoptimallypacked core, and are thus predisposed to undergo conformationalchanges on agonist binding, which have been proposed previouslyusing data from electron microscopy studies8,43. Notably, the buriedhydrophilic residues (Thr52 and Ser126) are highly conserved innAChRs, but are substituted by large hydrophobic residues (Phe, Leuor Val) in the nonchannel homolog AChBPs. These observationssuggest that the hydrophilic interior in nAChR has evolved forchannel specific functions. Indeed, our structure-guided mutagenesiscoupled with patch-clamp recording provides initial support forthis hypothesis.

In summary, our work complements previous structural studies ofthe AChBP homologs and the electron microscopic analyses of TorpedonAChR, substantially advancing our understanding of the structure-function relationships in the nAChR.

METHODSCrystallization, data collection, structure determination and analysis. The

extracellular domain of the mouse muscle nAChR a subunit (residues 1–211)

was prepared in yeast as described previously25. Because the wild-type protein

has limited solubility that is not suitable for crystallization, we screened a

library of PCR-generated mutants44. More than 2,000 colonies were randomly

selected for small-scale liquid culture. To identify mutants that were resistant to

aggregation and degradation, we harvested the culture supernatants, kept them

at 37 1C for 1 week, and then carried out 125I–a-Btx binding assays. One of the

mutants, a211, contained three substitutions (Val8Glu, Trp149Arg and

Val155Ala) and showed greatly improved expression and solubility. The a211

mutant was expressed in yeast Pichia pastoris as a secreted glycoprotein. a-Btx

was purchased from Invitrogen. The complex was prepared by mixing a-Btx

and a211 at a 1:1 M ratio and was further purified by gel filtration, as

monitored by multi-angle light scattering. The complex was concentrated to 8

mg ml–1 in a storage buffer, 150 mM NaCl and 20 mM HEPES, pH 7.5. Crystals

were grown by hanging drop at 4 1C using a reservoir buffer of 0.1 M sodium

citrate, pH 6.0, 18% 2-propanol and 8% PEG 4,000. Crystals belong

to the space group P212121 with cell dimensions a ¼ 42.498 A, b ¼ 85.68 A and

c ¼ 95.15 A. The complex crystals were stabilized in the harvest/cryoprotectant

buffer of 0.1 M sodium citrate, pH 6.0, 30% 2-propanol and 15% PEG 4,000,

and flash frozen with liquid nitrogen for cryocrystallography. The data

were collected at the ALS BL8.2.1 beam line at the Lawrence Berkeley National

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Laboratory. Data were reduced using DENZO and SCALEPACK45. Initial

phases were determined by molecular replacement using the coordinates

of Aplysia californica–AChBP (2BYP.pdb)13. Molecular replacement, refinement

and final analysis were done with CNS46. The crystallographic analysis statistics

are presented in Supplementary Table 1. The electron density map was of

excellent quality. All residues in a211 and a-Btx, including many ordered water

molecules, could be fitted into the density. Furthermore, a 10-residue

polysaccharide chain was built into the unambiguous density, where the

specific glycosidic linkages could be clearly defined. The final model has

88.4% residues in the most favored region, 11.2% in the additional allowed

region, and only one residue (0.4%) in the generously allowed region of

the Ramachandran plot. Structure illustrations were prepared using PyMol

(DeLano Scientific). Model building and structural comparisons were carried

out in O47. The coordinates for this crystal structure have been deposited in the

RCSB Protein Database under the accession code 2QC1.

Transfection, mutagenesis and patch-clamp recordings. The cDNA clones of

mouse a, b, d and e subunits (2:1:1:1 M ratio) in the vector pSM were

transfected in COS-7 cells using the Effectene transfection reagent (Qiagen). All

mutations were made using the QuikChange Site-Directed Mutagenesis kit

(Stratagene) and were confirmed by DNA sequencing. Whole-cell currents were

measured at B22 1C using the AxonPatch 200B Amplifier (Axon Instruments).

The pipette solution contained 140 mM CsCl, 10 mM EGTA, 10 mM HEPES,

pH 7.2, and 1 mM MgCl2. The resistance of the electrodes varied between

2.5–5 MO. The bath solution contained 135 mM NaCl, 5.4 mM KCL, 5 mM

HEPES, pH 7.2, 1.8 mM CaCl2 and 1 mM MgCl2. Whole-cell patches were

clamped at a membrane potential of –70 mV. ACh was applied extracellularly

using a Warner SF-77B fast perfusion system. Single-channel recordings were

done in the cell-attached configuration. Patch pipettes were pulled from

borosilicate capillaries, coated with Sylgard (Dow Corning) and fire polished

to a resistance of 6–10 MO. Dulbecco’s phosphate-buffered saline (DPBS:

137 mM NaCl, 0.9 mM CaCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2and 8.1 mM Na2HPO4, pH 7.4) was used in the bath. The pipette solution

contained DPBS and acetylcholine (ACh). The potential of the pipette was held

at +70 mV. Single-channel currents were recorded using an Axopatch 200B

amplifier. The data were digitized at a sampling rate of 100 kHz after low-pass

filtering to 20 kHz (8-pole Bessel). Analyses of the single channel data were

done using the QuB software suite as described48.

Deglycosylation, western blotting and Con A staining. COS cells expressing

the wild-type nAChRs were incubated with 100 unit ml–1 Endo H (New

England Biolabs) in DPBS for 20 min at 37 1C. The cells were rinsed twice in

DPBS before electrophysiological recordings were carried out. For western

blotting, surface proteins were labeled with biotin-7-NHS (Roche Diagnostics)

as described49, and extracted with streptavidin-sepharose beads. The receptor asubunit was detected by blotting with the monoclonal antibody mAb210

(ref. 50). To examine the efficiency of Endo H digestion, the cells were incubated

with rhodamine-conjugated concanavalin A (Con A, Vector Labs) in DPBS for

15 min at 4 1C, rinsed with DPBS, and visualized under a confocal microscope.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSThe authors thank Z. Hall for encouragement, J. Anglister for sharing resultsof the nuclear magnetic resonance experiments and advice, H. Lester andD. Dougherty for discussions, and X. Chen and E. Liman for critical reading ofthe manuscript. This research was supported by grants from Muscular DystrophyAssociation (Z.Z.W.) and the US National Institutes of Health (L.C. and Z.Z.W.).

AUTHOR CONTRIBUTIONSC.D.D. purified and crystallized the a211/a-Btx complex, collected X-raydiffraction data, solved and interpreted the structure, and wrote the manuscripttogether with L.C. Y.Y. carried out the mutational library screening, expressedand purified a211, and carried out functional studies of glycosylation, structure-guided mutagenesis, and patch-clamp recording. J.C.S. participated in thestructure refinement, analysis and manuscript writing. Z.Z.W. supervised theproject, conducted biochemical and pharmacological assays, electrophysiologicalrecordings, and data analyses, and wrote part of the manuscript. L.C. supervised

the project, collected X-ray diffraction data, solved and interpreted the structure,and wrote the manuscript together with C.D.D.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureneuroscience

Reprints and permissions information is available online at http://npg.nature.com/

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Erratum: Crystal structure of the extracellular domain of nAChR α1 bound to α-bungarotoxin at 1.94 Å resolutionCosma D Dellisanti, Yun Yao, James C Stroud, Zuo-Zhong Wang & Lin ChenNat. Neurosci. 10, 953–962 (2007); published online 22 July 2007; corrected after print 2 August 2007

In the version of this article initially published, the RCSB Protein Database accession code for the crystal structure was omitted. At the end of the first paragraph in the Methods section, the following sentence should have been included: “The coordinates for this crystal structure have been deposited in the RCSB Protein Database under the accession code 2QC1.” The error has been corrected in the HTML and PDF versions of the article.

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