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doi:10.1016/j.jmb.2004.12.031 J. Mol. Biol. (xxxx) xx, 1–23

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Refined Structure of the Nicotinic AcetylcholineReceptor at 4 A Resolution

Nigel Unwin

MRC Laboratory of MolecularBiology, Hills Road, CambridgeCB2 2QH, UK

0022-2836/$ - see front matter q 2004 E

Abbreviations used: ACh, acetylcbinding protein; MIR, main immunE-mail address of the author mas

We present a refined model of the membrane-associated Torpedo acetyl-choline (ACh) receptor at 4 A resolution. An improved experimentaldensity map was obtained from 342 electron images of helical tubes, andthe refined structure was derived to an R-factor of 36.7% (Rfree 37.9%) bystandard crystallographic methods, after placing the densities correspond-ing to a single molecule into an artificial unit cell. The agreement betweenexperimental and calculated phases along the helical layer-lines was usedto monitor progress in the refinement and to give an independent measureof the accuracy. The atomic model allowed a detailed description of thewhole receptor in the closed-channel form, including the ligand-bindingand intracellular domains, which have not previously been interpreted at achemical level. We confirm that the two ligand-binding a subunits have adifferent extended conformation from the three other subunits in the closedchannel, and identify several interactions on both pairs of subunitinterfaces, and within the a subunits, which may be responsible for their“distorted” structures. The ACh-coordinating amino acid side-chains of thea subunits are far apart in the closed channel, indicating that a localisedrearrangement, involving closure of loops B and C around the bound AChmolecule, occurs upon activation. A comparison of the structure of the asubunit with that of AChBP having ligand present, suggests how thelocalised rearrangement overcomes the distortions and initiates therotational movements associated with opening of the channel. Bothvestibules of the channel are strongly electronegative, providing a cation-stabilising environment at either entrance of the membrane pore. Access tothe pore on the intracellular side is further influenced by narrow lateralwindows, which would be expected to screen out electrostatically ions ofthe wrong charge and size.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: acetylcholine receptor; ion channel; refinement; electronmicroscopy

Introduction

The nicotinic ACh receptor is a member of thepentameric “Cys-loop” superfamily of transmitter-gated ion channels, which includes neuronal AChreceptors, GABAA receptors, 5-HT3 receptors andglycine receptors.1–5 The channel is found in highconcentrations at the nerve–muscle synapse, whereit mediates fast chemical transmission of electricalsignals in response to ACh released from the nerveterminal into the synaptic cleft. It is a large (290 kDa)

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holine; AChBP, ACh-ogenic [email protected]

glyco-protein, assembled from a ring of homo-logous subunits (a, g, a, b, d) and divided into threedomains: a large N-terminal extracellular ligand-binding domain, a membrane-spanning pore, and asmaller intracellular domain, giving it a total lengthof about 160 A normal to the membrane plane.The ligand-binding domain shapes a long, w20 Adiameter central vestibule and has two binding sitesfor ACh, which are about 40 A from the membranesurface on opposite sides of the pore. The poremakes a narrow water-filled path across themembrane and contains the gate, which openswhen ACh occupies both binding sites. Theintracellular domain shapes another, smaller vesti-bule, having narrow lateral openings for the ions.The receptor subunits in the ligand-binding

d.

Figure 1. Packing of receptors in the p2 tubular surfacelattice. (a) View down the axis of a single receptor and(b) view from the side, parallel with the membrane plane.Individual receptors are embedded in a curved lipidmatrix and come closest to each other at radial 2-fold axes(asterisks in (a)). A disulphide bridge between cysteineresidues of neighbouring d subunits lies at one such axis(blue asterisk); the C loops of neighbouring a subunits(ag) lie at the other (red asterisk). The direction of the tubeaxis and location of the membrane are indicated in (a) and(b), respectively. The cysteine residues at the 2-fold axisare the penultimate residues of the d subunits (seeFigure 5), and are in a region of weak densities wherethe polypeptide chain could not be traced (broken line in(a)). Individual subunits are in different colours (a, red; b,green; g, blue; d, light blue).

2 Refined Structure of Nicotinic Acetylcholine Receptor

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domain are each organised around two sets ofb-sheets packed into a curled b-sandwich andjoined through the disulphide bridge forming theCys loop, as was shown by the structure of theclosely related soluble protein, AChBP.6 The ACh-binding sites lie at the a–g and a–d subunitinterfaces, and are contributed mainly by residuesfrom loops A, B and C, connecting b-strands in thea subunits.7–9 The subunits in the membrane-spanning domain are each made from four a-helicalsegments (M1–M4).10 The helical segments arearranged symmetrically, forming an inner ring ofhelices (M2), which shape the pore, and an outershell of helices (M1, M3 and M4), which coil aroundeach other and shield the inner ring from the lipids.In the closed channel, the inner ring of helices cometogether near the middle of the membrane to makea constricting hydrophobic girdle, which consti-tutes an energetic barrier to ion permeation11,12 andmay function as the gate of the channel.10,13 Thesubunits in the intracellular domain each contributeone a-helix (part of the M3–M4 loop), whichtogether make the wall of the vestibule.14

Insight into the structural mechanism of gatinghas been obtained by electron microscopicalexperiments on helical tubes grown from Torpedopostsynaptic membranes,15,16 using a rapid spray-freezing technique to mimic the synaptic release ofACh and trap the open-channel form.17 Theseexperiments showed that binding of ACh initiatestwo interconnected events in the ligand-bindingdomain. One is a local disturbance in the region ofthe ACh-binding sites, and the other a larger-scaleconformational change, involving rotational move-ments predominantly in the two a subunits. Theinner M2 helices also change their configuration inresponse to ACh, widening the lumen of the pore atthe middle of the membrane. Higher resolutionstudies of the extended conformational change18

and of the structure in the membrane10 suggested asimplified mechanical model for the channel open-ing mechanism, whereby ACh triggers rotations ofthe inner b-sheets of the a subunits and the twistingmovement, communicated through the innerhelices, breaks the gate apart.

In addition to the structural details, summarisedabove, the roles played by individual amino acidresidues in determining the ligand-binding, gatingand cation-conduction properties of the AChreceptor have been extensively characterised bychemical labelling and by site-directed mutagenesisexperiments combined with electrophysiologicalstudy of function.19–28 Other experiments of thiskind, performed on GABAA, glycine, 5-HT3 andneuronal a7 receptors constitute a wealth ofcomplementary information.

We report here a preliminary three-dimensionalframework for relating these biochemical andphysiological data, based on refinement of a 4 Astructure obtained from electron images of thetubular Torpedo membranes frozen in a near-physiological ionic environment.10 The refinedmodel enables a detailed description of the whole

receptor in the closed-channel form, including theligand-binding region and vestibular entrances,which have not previously been interpreted at achemical level. We confirm that the two ligand-binding a subunits have a different extendedconformation from the three other subunits in theclosed-channel form of the receptor,18 and identify

Refined Structure of Nicotinic Acetylcholine Receptor 3

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several interactions at the subunit interfaces, andwithin the a subunits, which may be responsible fortheir “distorted” structures. The ACh-binding siteitself, which was not correctly identified in the 9 Amap,13 shows many features that are apparent inthe structure of AChBP. However, the organisationof the B and C loops at the binding site of the closedchannel differs from that in AChBP, where ligandis present, indicating that the binding reaction isaccompanied by a local structural rearrangement.A comparison of the two structures suggests howthe local rearrangement associated with AChbinding stabilises the alternative open-channelform of the receptor. Given our improved under-standing of this initial step, it is now possible tosketch a complete picture of the series of coordi-nated events leading to opening of the channel.Finally, we discuss the role of the vestibules, theionic surfaces of which create a strongly electro-negative environment at either entrance of thenarrow membrane pore.

Results

Structure refinement

The original 4 A data set was from 359 images oftubes,10 grown from Torpedo marmorata postsynapticmembranes.15 The tubes have four distinct helicalsymmetries, with individual molecules arranged ona p2 surface lattice15 such that the inside of the tubecorresponds to the inside of the cell.29 The receptorscome closest to each other near radial 2-fold axes(Figure 1(a)). A disulphide bridge between the dsubunits of neighbouring receptors30,31 lies near themembrane at one such axis; the a-subunit C loops ofneighbouring receptors lie w40 A from the mem-brane at the other (Figure 1(b)). We chose to refinethe receptor structure using standard crystallo-graphic methods, by neglecting these minimalinteractions and placing the experimental densitiescorresponding to a single molecule into an artificialunit cell (see Methods). To validate this approach,we monitored the agreement between the phasesalong layer-lines obtained by Fourier transform-ation of the images and the equivalent phasescalculated from model tubes. The helical phase

Table 1. Comparison between experimental and calculated p

Helical family

(K16,6) (K17,5) (K15,

62.19 66.17 67.8659.99 62.52 63.9458.79 62.02 63.7253.96 54.36 59.8653.57 54.61 57.92

Amplitude-weighted phase differences; resolution range, 100–4 A; nummodel: inner and outer sheets of ligand-binding domain, membrannoisy layer-lines from original data (see Methods). (3) Elimination of band domains; refinement of unit cell. (5) Molecular dynamics energyside-chains.

residual, comparing the experimental and calcu-lated terms, provided an independent objectivemeasure of the accuracy of the structure.A starting model of the receptor was built from

the coordinates of partial structures determined inearlier studies and from a-helical segments fitted tothe densities shaping the intracellular vestibule (seeMethods). Starting sets of phases were also calcu-lated from these coordinates after incorporatingthem into models of each of the four kinds of tube.More detailed coordinates were substituted later.While at first the coordinates and hence the modelswere incomplete and only approximate, reductionsin the crystallographic R-factors, paralleled bylower helical phase residuals, showed that themodels became more accurate as the refinementproceeded.As a preliminary step in the refinement, we used

the phases calculated from the model tube struc-tures as a reference to assess and optimise thequality of the original data set. The model-derivedphases provided a more sensitive test of the signalretained in the images at high resolution than theprevious reference, which had been derived solelyfrom the images. In this way, we found thatelimination from each helical family of a fractionof the layer-lines, the amplitudes along which weredominated by noise (see Methods), improvedthe quality of the data significantly (Table 1).Elimination of 17 “bad” images, for which theFourier phases showed no significant correlationwith calculated values at resolutions better than11 A also brought about some improvement(Table 1). It appeared that these images contributeddisproportionately large amplitude errors as aresult of overlap of terms along the layer-lines (seeMethods).The refinement of the structure was performed by

first treating the inner and outer b-sheet fragments,the membrane-spanning portion and the intracellu-lar a-helices of each subunit as separate rigid units.A major drop in Rfree and in the helical phaseresiduals (Table 1) was achieved by reducing the aand b unit cell dimensions (lying parallel with themembrane plane). The values of these parametersand the positional alignments were carefully opti-mised by several cycles of rigid-body refinement,minimising Rfree. The decrease in the a and b unit

hases along layer-lines during refinement

Steps

7) (K18,6)

64.06 (1)60.57 (2)60.15 (3)55.94 (4)56.16 (5)

ber of Fourier terms used for each estimate,w1.1!105. (1) Initiale-spanning domain, intracellular MA helices. (2) Elimination ofad images (17 out of 359). (4) Rigid-body refinement of fragmentsminimisation; modelling of loop regions; manual adjustments of

Table 2. Refinement statistics

P1 unit cell (refined) (A) aZ129.6, bZ129.6, cZ174.5Resolution range (A) 100–4Rcryst (%) 36.7Rfree (%) 37.9Fourier terms 95,988rms deviationsBond lengths (A) 0.016Bond angles (deg.) 2.16Average B-factor (A2) 67.2Ramachandran analysis (%)Most favoured 77.1Allowed 19.5Generously allowed 3.4Disallowed 0.0


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cell dimensions as a result of the optimisation was2.6%, implying that the averaged structure compos-ing the tubes was at a smaller radius than itappeared in the images. A minor degree offlattening probably contributed this effect, sincedeparture from a circular cross-section would causethe Fourier amplitudes to fall off more rapidly in theradial direction, a result suggested by the values

obtained for the overall anisotropic temperaturefactors (B11ZK21.3 A2; B22ZK21.3 A2; and B33Z42.6 A2, where B33 refers to this direction).

Additional improvements were obtained byseveral cycles of molecular dynamics refinement,using energy minimisation and backbone hydro-gen-bond restraints, followed by manual rebuildingand extension of loop regions, in O.32 The final R-factors were: RcrystZ36.7%; RfreeZ37.9% (Table 2).At these values the phase residuals had reachedtheir minimum values, implying that the bestaccuracy of structure, limited by the resolutionand by the amplitudes determined from images,had been achieved. The quality of the density maphad enabled placement of 80% of the 2335 aminoacid residues, the missing residues being locatedmostly in the M3–M4 intracellular loop, but also inthe b7–b8 loops of the non-a subunits, and the Ctermini (12 and 17 residues) of the g and d subunits.However, several of the loop regions were poorlydefined compared with the rest of the structure,making the tracing in these regions less reliable andprecluding detailed interpretation. The refinement

Figure 2. Examples of polypep-tide chains superimposed over thedensities in different regions of theexperimental density map. (a) Thisis a view of the “upper” part of thehelix M2 (b subunit) in the originalmap (PDB entry 1OED), and (b) isthe same region (including theb1–b2 loop) after refinement andimprovement of the density map(see Methods); the residues K46and E45 of the b1–b2 loop lie overthe M2–M3 linker, and D268 is acomponent of the extracellular ringof negative charge.21 (c) The mainimmunogenic region (MIR) of thea subunit next to d (ad) and theadjacent N-terminal a-helix, asviewed from the synaptic cleft; thelabelled residues W67–D71 contri-bute most to the antigenicity of theMIR (see Figure 5). (d) The helixMA (g subunit), shaping the intra-cellular vestibule of the channel, asviewed from the adjacent ag sub-unit; charged residues are labelled(see also Figures 7 and 8(b)). Con-tours are at 2s.

Figure 3. Ribbon diagrams of the whole receptor, as viewed (a) from the synaptic cleft and (b) parallel with themembrane plane. For clarity, only the ligand-binding domain is highlighted in (a) and only the front two subunits arehighlighted in (b) (a, red; b, green; g, blue; d, light blue). Also shown are the locations of aTrp149 (gold), the MIR and themembrane (horizontal bars; E, extracellular; I, intracellular). The dotted lines on the right denote the three main zones ofsubunit–subunit contacts. The apex of the C-loop of ad (broken trace in (a)) was not visible in the densities.


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led to relatively modest changes in membrane-spanning structure (rmsdCaZ0.8 A) compared withthe original model.10 The final refined modelshowed no residues (except for Gly) havingdihedral angles in the disallowed region of theRamachandran plot.

Three-dimensional density map

Examples of the polypeptide chains super-imposed on the experimental densities are givenin Figure 2. Figure 2(a) and (b) compare details of amembrane-spanning helical segment (M2 of the bsubunit) in (a) the original model, and (b) after therefinement. As is typical of the membrane-spanningregion, the positions of most of the side-chains havenot changed much as a result of the refinement; onthe other hand, the densities have improved, givingbetter definition for the bulky hydrophobic andextended side-chains. Thus we now have greaterconfidence that the assignments made in theoriginal study were correct, even if at the presentresolution the conformations of individual side-chains cannot be determined.

In a density map from a helical structure, thesignal-to-noise ratio may vary, depending on dis-tance from the helix axis: at high radius, high-orderBessel terms are mainly responsible for the densityvariations, and the retention of these terms dependson the accuracy of distortion corrections;33 at lowradius, the spatial overlap of different Bessel orders,which increases with resolution, becomes a

potential source of additional noise.14 However,we now find that the large side-chains are definedwith roughly equal clarity at either end of thestructure (Figure 2(c) and (d)), making it unlikelythat these effects have seriously compromised theoverall quality of the map.

Architecture and fold

The receptor is composed of elongated subunits,which associate with their long axes approximatelynormal to the membrane, creating a continuouswall around the central ion-conducting path. Thewhole assembly presents a rounded, nearly 5-foldsymmetric shape when viewed from the synapticcleft (Figure 3(a)), but is wedge-shaped whenviewed parallel with the membrane plane(Figure 3(b)).The subunits of the receptor all have a similar size

(maximum dimensions 30 A!40 A!160 A) andthe same three-dimensional fold. Figure 4 illustratesthis fold, as viewed in face-on and side-onorientations relative to the axis of the channel.Each subunit is a three-domain protein and sopartitions the channel naturally into its ligand-binding, membrane-spanning and intracellularparts. The N-terminal, extracellular portion is builtaround a b-sandwich core consisting of tenb-strands (inner sheets, blue; outer sheets, red)and contains one a-helix, like the protomer ofAChBP.6 This portion also contains several loopregions (e.g. the loops A, B and C, the Cys loop and

Figure 4. Ribbon diagrams of asingle subunit (a) viewed parallelwith the membrane plane, in orien-tations such that the central axis ofthe pentamer (vertical line) is (a)at the back and (b) to the side.The a-helices are in yellow; theb-strands composing the b-sand-wich are in blue (inner) and red(outer). Locations of the N and Ctermini, aTrp149, aV46, the Cys-loop disulphide bridge and themembrane (horizontal bars) areindicated. Part of the M3–M4 loop(connecting MA to M3) is missing.Labelling of secondary structuralelements and loops in this Figureand Figure 5 corresponds to thatgiven inpreviouspublications.6,10,34


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the b1–b2 loop), which are critical for receptorfunction. The membrane-spanning portion is com-posed of four a-helical segments, M1–M4, and thefunctionally important M1–M2 and M2–M3 loops.It is joined covalently to the extracellular domain atthe end of M1, and also interacts, through M2–M3,with the b1–b2 and Cys loops. The intracellularportion is composed mainly of the stretch ofsequence between M3 and M4, and includes acurved a-helix, MA,34 which precedes M4. Most ofthe rest of M3–M4 (i.e. M3–MA) appears to bedisordered and is not seen in the structure.

Figure 5 shows how the structural elements of thea polypeptide chain are organised in relation tothe amino acid sequence. The aligned b, g and dchains have the same organisation, and theircorresponding three-dimensional structures closelyresemble that of the a chain, except in someshort non-conserved regions (e.g. in the b8–b9 andC loops).

Symmetry

The approximate 5-fold symmetry of the

receptor was examined further by determining theangles required to achieve optimal least-squaressuperposition of the subunits around the pentamer.Deviations from 5-fold were found to be smallestin the membrane-spanning domain, where eachsubunit assumed an orientation lying within 28(s.d.Z1.618) of the value required for exact registerwith a 5-fold-averaged structure. These deviationsappeared to be a consequence of structuralvariations (which are most pronounced withM410) arising from the non-identical amino acidsequences. However, the deviations from 5-foldwere greater in the ligand-binding domain, becausethe two a subunits achieved exact register atrotation angles quite different from those of theother three. Using superpositions of the 190 mostclosely matched Ca atoms, for example, the devia-tions were: agZK3.178; bZC0.858; gZC2.718;dZC3.718; adZK4.088 (ag is the a subunit next tog; minus is anticlockwise, viewed from the synapticcleft). Hence there is an apparent anticlockwiserotation of the a subunits relative to the non-asubunits in the ligand-binding domain. Thisapparent rotation reflects the fact that the asubunits in the closed channel have a distinct

Figure 5. Aligned amino acid sequences of the four ACh receptor polypeptide chains. The sequences are fromT. marmorata, which differ in 48 places (cyan lettering) from those of T. californica (including the absence of the firstresidue of g). Locations of the MIR (critical segment in red), named loops, aTrp149 (star), and some key cysteine residues(green background) are indicated. Conserved residues forming the hydrophobic cores of the subunits in the ligand-binding domain and at the boundary between this domain and the membrane-spanning domain are shown with pinkand orange background, respectively. Elements of secondary structure, for the a subunits, are indicated above thesequences (yellow, a-helix; blue and red, b-strands composing the inner and outer sheets of the b-sandwich). The exactextents of the a-helices and b-strands are not accurately represented, given the limited resolution, but are similar for allfour polypeptides.


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conformation,17 associated with an alternativearrangement of the b-sheets.18 The different subunitconformations will be described in detail later.

Subunit–subunit interfaces

The main contacts between neighbouring sub-units occur at three levels in the structure (dottedlines, Figure 3(b)), and each of the interfacesexhibits similarly extensive subunit–subunit

interactions. Figure 6 tabulates the tentative inter-actions made between the a subunits and theadjacent b, g and d subunits, based on the estimatesof side-chain positions in the refined structure andassuming a cut-off distance for interacting atoms of3.9 A. We call the binding-site/disulphide-bridgeside of the subunit, theCside; and the other side,theK side. On theCside, residues of the B loop andinner sheet, M2, M3, M3–MA and MA interact withresidues of the inner sheet, M2, M1, M1–M2 and

Figure 6. Closely apposed residues at the interfaces ofthe a subunits with neighbouring b, g and d subunits. TheC symbol denotes the interface on the binding-site/disulphide-bridge side of the subunit (i.e. on theright face, Figure 4a); the K symbol denotes the interfaceon the distant side (left face, Figure 4(a)). Residues in eachcolumn are grouped according to their level in thestructure (Ca positions, beginning from the extracellularend) and categorised according to the structural elementsharbouring them. Charged residues are in bold font.


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MA of the g and d subunits. On theK side, residuesof the inner sheet, M2, M1, M1–M2 and MA interactwith residues of the A, B and C loops, M2, M3, M3–MA and MA of the b and g subunits.

The subunits in the ligand-binding domaininteract mainly through polar side-chains. Similarsets of interactions occur in AChBP,6 but they aremore extensive in the latter, consistent with the factthat AChBP (in the presence of ligand) has a smallerradius of gyration (29.8 A compared with 31.8 A forthe ligand-binding domain, calculated from theatomic coordinates), and so is more compact. It isnotable that the interfaces on both sides of the asubunits contain charged side-chains, which formprobable ion pairs with side-chains on neighbour-ing subunits (aR79 with bD155, gE154; aD152 withgR78, dR81). These interactions may be important in

stabilising the resting, closed-channel conformationof the a subunits. There are no equivalent pairingsbetween charged side-chains at the non-a, b–dinterface.

In the domain shaping the membrane pore,hydrophobic side-chains projecting from the helicesM1, M2 and M3 are mainly responsible for thesubunit–subunit contacts. A probable exception isan interaction involving positively charged side-chains on d, g (R277, K271) with the side-chainsE262 of the a subunits composing the pore-lining“extracellular ring”.21 The subunit–subunit contactsbetween the helices occur predominantly in theintracellular leaflet of the bilayer, and implicaterelatively few residues on M1 and M3 (Figure 6). Atthe intracellular face of the membrane-spanningdomain, and at the extreme intracellular end of thereceptor, there are several additional subunit–subunit contacts implicating the M1–M2 loop, theM3–MA loop and MA. However, the description ofthese regions may be incomplete, given that parts ofthe M3–MA loop may be involved that are notvisible in the structure.

The narrow interstitial spaces between the con-tact areas on the subunit interfaces are of specialinterest because they provide pathways (or poten-tial pathways) for diffusing ions. Most of them arelined by polar or negatively charged side-chainsand would therefore be selectively permeable tocations. They occur on both sides of the membraneclose to the membrane surfaces. However, thelargest open spaces are on the intracellular side,between neighbouring MA helices, and the surfacesframing each of these contain several negativecharges (Figure 7).

The intracellular MA helices from each subunittogether create an inverted pentagonal cone havingfive intervening open spaces, or windows, ofsimilar size. These windows represent obligatoryion pathways, since no alternative routes exist fortransport into, or out of the intracellular vestibule.The windows have a maximum width of onlyw8 A, which is comparable with the diameter of asodium or potassium ion surrounded by its firsthydration shell. The windows would thereforeforce direct interaction of the hydrated ion withtheir negatively charged surfaces, facilitating cationtransport while electrostatically repelling anionsand preventing large ions from going through.

The vestibules

The extracellular and the intracellular vestibulesare both narrow enough (w20 A wide) to ensurethat charged groups lining their surfaces wouldinteract electrostatically with the passing ions, yetwide enough not to necessitate direct contact, whichcould slow their movement.11 Again, the chargedgroups are almost entirely of negative polarity(Figure 8(a) and (b)). The resulting cation-stabilisingenvironments would increase the concentration ofcations relative to that of anions at both entrances ofthe narrow membrane pore, and so promote

Figure 7. Electrostatic potential surface representations showing entry/exit windows for cations between the MAhelices of different subunits on the intracellular side of the membrane. The windows formed between d and b, andbetween g and ad, are on the left and right, respectively. Labels identify exposed charged side-chains. The location of theintracellular membrane surface is indicated (horizontal bars). The sphere in the d–b window is the size of a potassiumion (2.7 A diameter). Portions of the polypeptide chain other than MA are involved in shaping the upper portions of thewindows; the stretches in the Figure, and in Figure 8(b), are from: a233–a248 (including the M1–M2 loop); a295–a306(including the C terminus of M3); a374–a411 (including the N terminus of M4). The electrostatic surface was contouredbetween K15kT/e and C15kT/e; negative and positive charge in red and blue, respectively.


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efficient bidirectional transport of cations throughthe open channel. As will be discussed later, it islikely that the vestibules (including the intracellularwindows) contribute significantly to the chargeselectivity of the channel, and that the “selectivityfilter” is not just a local region, identified bymutation experiments, at the intracellular end ofthe pore.

Interactions across subunit domains

Several components of the ligand-bindingdomain could in principle be involved in commu-nicating the ACh-triggered conformational changeto the membrane-spanning domain, where the gateis located. But the structure (Figure 4) shows thatonly the Cys loop, the b1–b2 loop and the polypep-tide chain (through the covalent connection) makedirect contact. The two loops interact with thestretch of amino acid residues M2–M3, linking theM2 and M3 helical segments: the Cys loop bystraddling M2–M3 near the N terminus of M3, andthe b1–b2 loop by straddling M2–M3 near the Cterminus of M2. It is notable that the Cys and b1–b2loops are not in equivalent locations relative to themembrane-spanning domain for all subunitsaround the pentamer (Figure 9(a)). In the a subunits(which have a special conformation; see below) theyare slightly displaced along M2–M3, bringing theb1–b2 loop 2–3A closer to the axis of the channel.

The interaction of these loops with M2–M3involves different amino acid residues, dependingon the subunit in question and, in the case of theCys loop, implicates additional residues near the

ends of M1 and M4. However, one set of inter-actions, involving the consecutive residues FPF, ofthe Cys loop, and the residues I, Y and F (aligningwith aI274, aY277 and aF280) at the end of M3, iscommon to all five subunits. The FPF residuesproject downward from the extremity of the Cysloop towards the membrane to meet the I, Y and Fresidues extending upward from M3 towards theligand-binding domain (Figure 9(b)). These sixresidues are highlighted in Figure 5 (orange back-ground): together they form a cluster, the hydro-phobic and predominantly aromatic character ofwhich is conserved throughout the receptor super-family. The flexible aromatic residues may serve toaccommodate movements that occur near the endof M3 during gating (see below), as well as toanchor the Cys loop to the membrane-spanningdomain.Two residues of the b1–b2 loop appear to be

important in forming interactions with M2–M3: theresidue aligning with aV46 and the adjacentglutamic acid residue (aligning with aE45), whichis conserved throughout the superfamily. Theirside-chains together make an arc embracing theM2–M3 backbone, and may therefore help to fix theend of M2, which is not in contact with the othermembrane-spanning helices at this level in thestructure. The aV46 side-chain (V44 in AChBP) fitsinto a hydrophobic pocket made by the endresidues of M2,10 consolidating the clasp. However,the side-chains equivalent to aV46 in the othersubunits seem slightly displaced from the ends ofthe M2 helices (Figure 9(a)) and do not makeequivalent contacts: for example, the lysine residue

Figure 8. Central sections showing the inner surfaces of(a) the extracellular and (b) the intracellular vestibules.Both vestibules are lined by an excess of negativelycharged groups, promoting a cation-stabilising electro-static environment. (a) A view looking towards the ag–bsubunit–subunit interface; (b) a view looking towards theag subunit, with g and b on either side. Labels identifyexposed charged side-chains. The locations of themembrane pore (arrows) and the membrane surfaces(horizontal bars) are indicated. Electrostatic surfaces arecontoured as in Figure 7.


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on the b subunit (bK46) overlies M2–M3, with theterminal amino group apparently exposed tosolvent (Figure 2(b)).

Conformations of individual subunits

The conformations of the subunits in the closedchannel were investigated in an earlier study18 by

dividing the b-sandwich core of the AChBP proto-mer into the inner and outer b-sheet fragments, andfitting these fragments as rigid bodies to a 4.6 Aresolution density map. This study had suggestedthat to a first approximation there are two alterna-tive arrangements of the sheets related by rotationsabout the Cys-loop disulphide bridge: one arrange-ment characteristic of the two a subunits and theother characteristic of the three non-a subunits.However, the relatively poor quality of the 4.6 Amap, omission of loop regions and the strict rigid-body approach limited the conclusions drawn. Therefined, energy-minimised structure, describedhere, allowed a more accurate assessment of thealternative conformations of the subunits.

To conduct an initial comparison, we rotated eachsubunit by a multiple of 728 about an axis normal tothe membrane plane to bring it into 5-fold registerwith a reference subunit. We then aligned ittranslationally to superimpose the midpoints ofthe lines connecting the Ca atoms of the pair ofcysteine residues forming the disulphide bridge.This comparison confirmed that the inner sheets ofboth a subunits were rotated anticlockwise relativeto those of the other subunits, when viewed fromthe synaptic cleft (curved arrow, Figure 10(a)). Theindividual Ca traces showed systematic differences,distinct from differences which could arise fromsimilar, but non-identical subunit structures or frominaccuracies in chain tracing. As before, the rotationaxis of the inner sheets was normal to themembrane and in the vicinity of the disulphidebridge. However, to achieve pairwise least-squaressuperpositions of the inner sheets onto ag, theangles were: adZC1.68; bZC10.98; gZC11.28;dZC11.78. That is, the measured rotation anglesof the a chains, relative to the non-a chains, wereabout 58 smaller than the values (w158) estimatedpreviously.

While the anticlockwise rotations in the asubunits applied to the entire set of inner b-strands,including the loop (b2–b3) harbouring the mainimmunogenic region (MIR), the adjacent N-term-inal a-helices were not significantly rotated, butretained orientations similar to those of the non-asubunits. As a result, the a subunits have theN-terminal helix and the MIR separated by a cleft(Figure 2(c)). The wider separation between thesetwo regions in the a subunits may reflect smalldifferences comparedwith the non-a subunits in theset of interactions that hold the N-terminal helix inplace. This helix is not tightly associated with thebody of the subunit as it is in AChBP.

We also confirmed the previous finding thatthe strands composing the outer sheet of theb-sandwich tilt more steeply in the case of the athan the non-a subunits, when viewed from adirection parallel with the membrane plane (Figure10(b)). However, the differences in tilt of theindividual strands (b9, b10, b7 and b4) was notuniform, since the strand b9 of the a subunits runsalmost parallel with those of the non-a subunits,and the strand b10 is intermediate between the two

Figure 9. Interaction of loops at the boundary between the ligand-binding and membrane-spanning domains. (a) Thisis a view down the central axis of the receptor showing, for each subunit, the locations of the Cys loop (a128–a142) andthe b1–b2 loop (extended to include a41–a50) in relation to the underlying domain forming the membrane pore; theasterisk and arc on both a subunits indicates the position of the rotation axis relating the inner sheets (see Figure 11(a)).(b) This is a view of the domain boundary (a subunit) from the side, showing the Cys loop and b1–b2 loop in relation toM2–M3 (dotted trace), and the locations of residues mentioned in the text; the large dot at theM3 end of theM2–M3 tracedenotes a conserved glycine residue (aG275), which may provide a point of flexure during gating. The Cys loop, the b1–b2 loop, the extension of M1 into strand b10 of the ligand-binding domain, and components of the membrane-spanningdomain are in blue, red, green and grey, respectively.

Figure 10. Superposition of backbone Ca traces of portions of the subunits after they have been rotated by multiples of728 to bring them into 5-fold register and aligned on the Cys-loop disulphide bridge. The slabs shown lie (a) parallel withand (b) obliquely to the membrane plane. (a) A cross-section through the subunits at the level of the disulphide bridge(S–S), as viewed from the synaptic cleft; the curved arrow denotes the anticlockwise rotation of the inner b-strands of thea subunits, relative to those of the non-a subunits; the short arrow denotes accommodating displacements of the outerstrands, b9. (b) The four outer b-strands, as viewed from the external surroundings. The inner and outer b-strands haveblue and red labels, respectively; the trace colours are: a, black; b, pink; g, red; d, purple.


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Figure 11. Comparison of a and non-a subunits in the ligand-binding domain, as in Figure 10, after translationalalignment of the inner b-sheets to a common rotation axis (asterisk). (a) The projected backbone traces of the five sets ofinner sheets (including the b1–b2 loops and the equivalent region of AChBP), as viewed from the synaptic cleft. (b) Astereo view (from the direction of the oblique arrow in (a)) comparing a with a non-a subunit (g) over the wholeb-sandwich domain; inner and outer b-strands are identified with blue and red labels, respectively; the arrows near thebottom of the Figure denote displacements of b-strands in a relative to non-a (see also Figure 10(a)). The colours are: a,black; b, pink; g, red; d, purple; AChBP, green.


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extremes. These disparities reflect a small differencein twist between the b9–b10 hairpins of the a andnon-a subunits (w108 over 20 A). The b9–b10hairpin of the a subunit is less twisted, diminishingthe overall (right-handed) twist of the outer sheet.The “untwisting” causes the b9 strand of a to bedisplaced outwards at the level of the disulphidebridge (short arrow, Figure 10(a)), creating the space

that is needed to accommodate the anticlockwise-rotated inner sheet.

Refinement of rotation axis

It had been proposed that the a subunits in theclosed channel were in a “distorted” configurationrelative to the others and that the conformational

Figure 12. Interpretation of the ACh-binding region ofthe closed channel at the interface between the a and gsubunits, showing the loops B and C (a subunit), theadjacent strands, b5 and b6 (g subunit) and the attachedamino acid side-chains. The slab is of the upper part of theACh-binding region, viewed from the synaptic cleft.Some key residues implicated in ACh binding arelabelled. The Ca backbone and side-chains are in red (a)and blue (g). As indicated, a salt-bridge between aD152and gR78 may be involved in stabilising the B loop. Thesuperimposed experimental densities show weak fea-tures associated with the C loop; contours at 2.0s (grey)and 3.0s (cyan).


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change to open the channel involved movementsthat brought them into a configuration similar tothat of the non-a subunits, making the wholeassembly more symmetrical.18 The rotational move-ments of the inner sheets played a key role in thismechanism, because of their close association(through the loop b1–b2) with the pore-lining M2helices. It is therefore of interest to establish thelocation of the true axis of rotation within theprotein subunit. This does not have to pass exactlythrough the disulphide bridge, provided that thecysteine residue (aC128) associated with the innerstrand (b6) and the cysteine residue (aC142)associated with the outer strand (b7) undergosimilar small displacements.

To define a common axis, we determined thetranslations that minimised the rms deviationsbetween the Ca coordinates of the five sets ofinner sheets. Following alignment by this criterion,the sheets superimposed tightly on the same semi-circular arc (Figure 11(a)), with the a subunitsclearly belonging to one group and the non-asubunits to another (values of rmsdCa with respectto ag were: adZ0.7 A; bZ2.1 A; gZ2.2 A anddZ2.2 A). Pairwise least-squares analyses of differ-ent a and non-a combinations then yielded similarlocations for the rotation axis, which were centred8–9 A from the midpoint position relating to thedisulphide bridge (Figure 10(a)). We interpret theline passing through this point (Figure 11(a)), andnormal to the membrane, to be the best estimate ofthe true axis of rotation. The line extends throughthe hydrophobic core of the ligand-binding domain,the base of the b9–b10 hairpin and between helicesM1, M3 and M4, forming the outer protein shell ofthe membrane-spanning domain (Figure 9(a)).

Figure 11(b) compares the a subunit with a non-asubunit (g), after this alignment, in the context ofthe whole b-sandwich domain. As indicated inFigure 10, the anticlockwise-rotated strands of the ainner sheet (grey arrow) are complemented by anoutward displacement of the strand b9 of the outersheet (black arrow), near the “bottom” of thisdomain.

ACh-binding region

The b9–b10 hairpin of the a subunits incorporatesthe C loop, which is implicated in ACh binding. TheC loop is resolved only weakly in both a subunits,suggesting it is flexible in the absence of ACh.Densities are not visible for residues 191–194 in ad,but are present in the equivalent part of ag, possiblybecause in the crystal lattice the C loop of ag is nextto the C loop of an adjacent subunit (see Figure 1),which stabilises its conformation. Figure 12 showsthe C loop of the ag subunit and the neighbouringregion, with the polypeptide backbones and inter-preted side-chain positions superimposed on theexperimental densities. The labelled side-chainsY190, Y198, C192 of the C-loop and W149 of the Bloop are conserved in ACh receptor a subunits andco-ordinate to the bound ACh analogue, carbamyl-

choline, in the complex with AChBP.9 Thus thiswhole region, including the A loop (not visible inthe Figure) and the adjacent strands b5 and b6 of theg (or d) subunit, is involved in shaping the ACh-binding pocket of the receptor.In lower resolution studies of the ACh receptor,13,

14 we had found that the a subunits differed mostfrom the non-a subunits, not at the interface regionshown in Figure 12, but closer to the centre of theb-sandwich. Both a subunits displayed weakerdensities at the centre of the sandwich, indicatingthat they had a more open structure there than didthe other subunits, so we had identified the centralregion as the “putative ACh-binding site”. It isevident now from the refined structure that themore open appearance is a consequence of thedistinct inner- and outer-sheet arrangements inthe a subunits. The b9–b10 hairpin of the a subunits,for example, is w1.5 A further from the oppositely


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facing inner b2 and b6 strands than it is in the othersubunits at the level of the binding sites. Clearly, theearlier identification was mistaken, and the actualbinding sites are closer to the g and d-subunitinterfaces in locations equivalent to those inAChBP.6,9

As Figure 12 shows, the C loop projects awayfrom the body of the a subunit in the closed-channelform of the receptor. This is in contrast with (ligand-bound) AChBP, where the C loop projects approxi-mately tangential to the central 5-fold axis,9 and sois closer to the A and B-loop residues implicated inACh binding. The Ca distance between C192 (Cloop) and W149 (B loop), for example, is 12 A inAChBP, compared with 18 A in the receptor. Theseloops must therefore undergo quite large relativemovements in order to allow coordination of thebinding residues to the ACh molecule: a conclusionconsistent with biochemical results implying thatthe C-loop cysteine residues (C192, C193) move in afew angstrom units toward a negative subsite whenagonist binds.35

The involvement of the B loop in this localrearrangement is likely to be critical because itjoins the outer to the inner b-sheets and thereforemust participate directly in effecting their relativedisplacements, which leads to opening of thechannel (see Discussion). The B loops of the asubunits come close to the inner b5 and b6 strandsof the g and d subunits and so may be stabilised inthe closed channel by interactions across thesubunit interface. One example is a possible salt-bridge between aD152 and gR78 or dR81 (Figure12), but there are several other potential contactswith the g or d subunits in this region (Figure 6).

Comparison with AChBP

Each of the subunits in the ligand-bindingdomain has a hydrophobic core of conservedresidues which are grouped into three clusters, asin the protomer of AChBP.6 The core residues of thereceptor (pink background, Figure 5) are equivalentto those identified in AChBP, with the exceptionof the leucine (aL6) near the end of the N-terminala-helix. Several hydrophobic residues not identifiedin AChBP also contribute to the core in the receptor.These residues are conserved among the nicotinicsubunits and align with aV33, aL56, aI78, aL80,aL108, aW118 and aF124. The overall matchingfollows the predicted pattern of inward and out-ward-facing residues,6,36 confirming that the corethree-dimensional structures of all the pentamericsubunits are essentially the same.

Furthermore most of the surface loops correlateclosely between the two structures, even in regionswhere the amino acid sequences are not conserved.The hydrophobic Cys loop of the receptor, forexample, has a similar fold to its hydrophiliccounterpart in AChBP, but for the insertion of theextra residue in the receptor. The insertion extendsthe “heel” on the foot-shaped loop (Figure 4(a)), sothat the loop straddles more completely the M2–M3

linker of the membrane-spanning domain. How-ever, the stretch b5–b5 0 (aligning with aV103–aM105) of the ligand-binding domain is signifi-cantly different from its counterpart in AChBP,folding inwards toward the core of the subunit. Thebinding site residue aW149 is thereby exposedmore fully to the lumen of the vestibule (Figure3(a)). Portions of the b8–b9 loops of the b, g and dsubunits have no counterpart in AChBP, but theseregions are also missing from the receptor structure.

AChBP does not have an equivalent of the MIR,a special region at the extreme extracellular end ofthe a subunits37 that constitutes the major bindingsite for antibodies in the auto-immune disease,myasthenia gravis.38,39 The critical segment of theepitope of these antibodies has been localised toresidues aW67–aD71 (red letters, Figure 5), withaN68 and aD71 contributing most to the anti-genicity.39,40 This five-residue segment forms a loopapparently having the same b-folded structure(Figure 2(c)) as was found in NMR studies ofpeptide–antibody complexes.41 All five residues(including the conserved core residue aW67)appear to be exposed to solvent because ofthe wide separation between the loop and theN-terminal helix. The equivalent loops of the othersubunits have the same fold, but are closer to theirrespective helices (Figure 11(b)), which at leastpartly bury the inward-facing residues aligningwith aW67 and aD71.

Discussion

The refined 4 A structure reported here providesa chemical interpretation of all the main functionalregions of the ACh receptor, as they would appearunder near-physiological ionic conditions in Torpedopostsynaptic membranes. Although the final crys-tallographic R-factor was only 36.7%, limited by thequality of the amplitudes from images, we demon-strated that the polypeptide chains could now betraced with reasonable confidence over the entirelength of the molecule (Figures 2 and 12). Thisincluded the extreme extracellular and intracellularends (at high and low tube radius), which were theparts most affected by errors inherent in the helicalanalysis. It was also possible to use comparisonsbetween the experimental phases and the phasesfrom calculated helical structures (Table 1) toidentify and correct for deficiencies in the originaldata and to validate the agreement between theatomic model and the experimental density map.Such comparisons were not feasible in the lowerresolution investigations of the whole molecule.The present interpretation is therefore both moreaccurate and more detailed than previously, andshould furnish a preliminary three-dimensionalframework to guide our understanding of thefunctional properties of this ion channel and ofothers in the superfamily.

In a recent structural analysis of the membrane-spanning portion of the receptor, we described the


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arrangement of a-helical segments encircling the(closed) ion-conducting pore,10 and suggested howthe extended conformational change initiated byACh opens the gate of the channel throughrotational movements communicated along thepore-lining helices. The present study complementsthat analysis focused on the gate and the membranepore, providing now a description of the structurearound the ACh-binding site. The binding site inthe closed channel differs considerably from that inAChBP, where ligand is present, indicating thatthe binding reaction is accompanied by a localisedstructural rearrangement. Comparison with AChBPshows that the localised changes blend naturallywith the larger-scale structural changes, yielding asimple explanation (discussed below) for how thebinding reaction is used to drive the extendedconformational change. Given the improved under-standing of this initial step, it is now possible tosketch a complete picture of the series of coordi-nated events leading to opening of the channel.

Special conformation of the a subunits

Our analysis of the ligand-binding domainconfirmed that the subunits of the closed channelhave two alternative extended conformations: onecharacteristic of either a subunit, and the othercharacteristic of the three non-a subunits.18 How-ever, the refined structure enabled more accuratemeasurement of their differences. We determinedthat the inner sheets of the b-sandwich composingthis domain are rotated by approximately 108in both a subunits relative to the non-a subunits(i.e. about 58 less than estimated earlier), about anaxis normal to the membrane plane. We determinedthat the rotation axis lies 8–9 A from the Cys-loopdisulphide bridge, so that it passes almost centrallybetween the ends of helices M1, M3 and M4forming the outer protein shell of the membrane-spanning domain (Figure 9(a)). We also observed,as before, that the outer sheets of the b-sandwichhave slightly different orientations in the a com-pared with the non-a subunits (Figure 10(c)), andwere able to show in this study that the b9–b10portion of the a outer sheets has a reduced twist.

Several interactions across the subunit–subunitinterfaces were identified that might be involvedin stabilising the special conformation of the asubunits. These occur on both sides of both asubunits and implicate residues on the B loop aswell as on the inner b-sheets. Salt-bridges are likelyto be important, since they are present only atinterfaces made with the a subunits. The“untwisted” configuration of the b9–b10 hairpinmay be stabilized through an additional set ofinteractions unique to the a subunits: possiblythrough a salt-bridge between D200 on b9–b10and K145 on b7 at one end, and a hydrophobiccontact between aI210 on b9–b10 and the Cys loopat the other end (see Figure 9(b)). Since these interand intra-subunit interactions are similar, or thesame, for both a subunits, one would expect their

three-dimensional structures to be the same. Withthe exception of differences at the binding site(where the C loop of ad is disordered), and ofvariations in side-chain conformations (whichcould not be resolved), this seemed to be the case.

Local rearrangement associated with AChbinding

We call the special conformation of the a subunitsa “distorted” conformation because these subunitsconvert to a conformation similar to that of the non-a form, and of (ligand-bound) AChBP, when thechannel is opened by ACh.18 It is as if the a subunitsare held initially in a distorted (or tense) state, bythe interactions just described, and the energy ofbinding overcomes the distortions, allowing the asubunits to convert toward the (relaxed) non-a formthat they would have if these interactions did notexist (in analogy with other allosteric proteins).42 Bycomparing the binding site region of the a subunit(having no ligand present) with the correspondingregion of AChBP (having ligand bound) we canobtain insight into how the local disturbanceassociated with the binding reaction drives themovements that overcome the distortions, allowingthe a subunits to relax. AChBP provides a closeanalogue of the binding site in the receptor,43

although the C loop of the a subunit has an extraresidue at P197. In particular, both proteins containthe same set of aromatic residues, which arrange ina tight “box” around the quaternary ammoniumgroup of the bound molecule.9

Figure 13(a) and (b) give simplified represen-tations of the binding-site region shown in Figure 12and the equivalent region of AChBP to which theACh analogue, carbamylcholine is bound.9 Com-parison of these free and ligand-bound structuresshows that the B and the C loops would both closein around the ACh molecule to enable coordinationof the relevant side-chains: the B loop by rotatingclockwise (large arrow) and the C loop by a twistingand rotating movement (small arrows). The A loop(not shown) would also be involved, but theindicated movements are smaller.The AChBP protomer can be aligned translation-

ally to the rotation axis relating the inner b-sheets ofthe receptor, after optimal superposition of thepentamers, and thereby identified unequivocallywith the non-a conformation (Figure 11(a)). Such analignment also allows a realistic superposition ofthe protomer with the a subunit to simulate how thelocal rearrangement would be communicated tothe b1–b2 loop (and from there to the gate in themembrane). As Figure 13(c) shows, the B loop isjoined to strand b8 of the inner sheet, and the innersheet itself would act as a rigid connecting link,transmitting the clockwise rotation initiated at the Bloop to the b1–b2 loop on the opposite face of thesubunit. Alternatively the change (pink to green)can be regarded as a conversion from the distortedto the (relaxed) non-a form of the subunit. Theimplication is therefore that the ACh-binding

Figure 13. Comparison of the ACh-binding region inthe a subunit with ligand-bound AChBP9 suggests howthe local rearrangement caused by ACh initiates theextended conformational change to open the channel. TheFigure shows: (a) simplified Ca traces of the ligand-binding region of the a subunit, with labels identifyingparts mentioned in the text; (b) the equivalent region ofAChBP complexed with carbamylcholine (PDB entry1UV6); (c) the two regions superimposed after alignmentto a common rotation axis (Figure 11(a)), and extension toinclude the two b1–b2 loops, which are connected to the Bloops through the inner sheets (arcs). The a subunit is inthe closed-channel conformation, whereas AChBP is ananalogue of the open or desensitised state.76 Closure ofthe B and C loops around the bound agonist changes theorientation of the B loop (large arrow in (b)) and twist of


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reaction would drive the movements to open thechannel by initiating a local rearrangement thatmakes the non-a conformation more stable.

Several of the residues participating in the localrearrangement may play key roles in enabling thelarger scale movements, and hence in couplingthe binding reaction to opening of the channel. Theglycine residue aG153 (Figure 13(a)), for example,may help in conferring flexibility on the B loop aswould be needed to enable the linkedmovements ofthe inner sheets. The mutation aG153S is in fact anaturally occurring mutation, causing a slowchannel congenital myasthenic syndrome in whichthe channel reopens more readily than in the wild-type.44,45 The aspartic acid residue aD200 (Figure13(a)), through interaction with strand b7, may helpin constraining the twist of the b9–b10 hairpin ineither of the alternative conformations. Themutation aD200N is another well-characterisedmutation that leads to impaired gating of thechannel.46,47 Mutations of the A, B and C-loopresidues aY93, aW149, aY190 and aY198, whichinteract directly with bound ligand in AChBP,9 alsoaffect gating.48–51

Propagation of the conformational change

The conversion of the a subunits to a non-a-likeconformation is the major extended conformationalchange controlling channel opening, according tothe structural differences revealed in receptorsbriefly exposed to ACh.17,18 As analysed here, thetransition would involve mainly rigid-body move-ments of the inner and outer-sheet parts of theb-sandwich and small rearrangements or readjust-ments by the connecting loops. The inner sheetwould be the primary structural element determin-ing the gating function of the channel, making useof a rotational movement to effect a displacementof the b1–b2 loop next to the helix lining themembrane pore; whereas the outer sheet wouldprovide the structural framework needed to initiatethe rotational movement and to accommodate thedisplacements involved. At the same time, disturb-ance of the neighbouring subunits would beminimised by having the rotation axis normal tothe membrane plane.

The twist of the a-subunit b9–b10 hairpin seemslikely to play an important role in coordinatingthese movements. We showed that it is “untwisted”in the closed channel, creating room for the innersheet to be in the rotated-anticlockwise position(Figure 10(a)). In the open-channel, it would be“twisted”, as in a non-a subunit, fitting (togetherwith the b8–b9 loop) against the inner sheet in therotated-clockwise position. Because of the comple-mentary nature of changes such as these, the

the b9–b10-hairpin (twisted arrow in (b)). It is proposedthat these changes together drive the clockwise rotationsof the inner sheets, favouring the open-channel extendedconformation.


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relative displacements between the inner and outerparts of the b-sandwich, in the interior of thesubunit, would be quite small. In fact, no funda-mental difference was detected between the a andnon-a subunits in their interior organisation,suggesting that the relative displacements wouldbe accommodated through minor adjustments intorsion angles of the inward-facing side-chains.

Coupling to the membrane

How do the rotational movements in the ligand-binding domain communicate through the M2helices to open the channel? The limited resolutionof this study, the absence of a high-resolutionstructure of the open channel and the limitedinformation available from mutagenesis studies52,53 do not yet allow a definitive answer. However,the structure suggests that the b1–b2 and the Cysloops may together influence M2–M3 at either endsto control the gating movements. The b8–b9 loop isalso implicated54–56 (Figure 10(a)), but it does notextend to the membrane-spanning domain.

One possibility is that the fast gating of the M2helices, in the absence of a ligand-binding domain,would be analogous to that of M2 peptides in lipidbilayers, which open and close constitutively likethe authentic channels,57 and that these motions aredisallowed by the a subunits in their closed-channelconformation. The b1–b2 and Cys loops are 2–3 Acloser to the end of M2, along the M2–M3 linker,in the a than in the non-a subunits (Figure 9(a)). Inthese locations they might respectively lock M2 inits closed configuration and restrict the flexure ofM2–M3 conferred by the conserved glycine residue(aG275) at the end of M3 (Figure 9(b)). When AChbinds and the loops rotate back toward their non-alocations, these restrictions could be relieved,allowing the fast gating motions to occur.

A displacement of the two loops over M2–M3 isconsistent with the change in accessibility duringgating of the residue a1A284 of the GABAA

receptor,58 since this residue (aligning withaL2731) lies between the b1–b2 and Cys loops onM2–M3. However, an alternative possibility is thatthe specific interaction involving aV46 on the b1–b2loop is maintained, and that the loop movementpromotes opening of the pore by drawing the end ofM2 away from the axis of the channel. Whateverthe precise nature of the coupling, the fast gatingkinetics implies that the activation energy requiredto switch between the open and closed pores is verysmall. Only a minor structural perturbation shouldtherefore be sufficient to tip the balance one way orthe other.

Coordinated gating movements

The opening and closing of the ACh receptorchannel is usually regarded as a concerted process,whereby the whole protein switches rapidlybetween alternative “pre-existing” conformations.59

It may also be considered in terms of a

“conformational wave”.60 However, it is betterunderstood mechanistically if the action is brokendown into a series of coordinated events. Thestructural details described above, and previousresults focusing on the membrane pore,10 nowprovide a complete (simplified) model for the seriesof coordinated events leading to opening of thechannel. The steps would be: (a) ACh enters the twobinding sites, causing loops B and C of the asubunits to close in around the bound molecule;(b) the resulting local rearrangement reduces thestability of distorted form of the a subunits infavour of the relaxed (non-a) form; (c) the extendedconformational change is therefore initiated, dis-placing the b1–b2 loops of a over the ends of theirrespective M2 helices; (d) the displacements unlockthe interactions that restrict the rotational move-ments of the pore-lining helices; (e) the helicesmove, destabilising the weak hydrophobic inter-actions holding the gate together, so that it breaksapart.Our results imply the a subunits are the principal

mediators of the conformational change that opens(or closes) the channel, whereas the other subunitscontribute critically in influencing ACh binding andin stabilising closed-channel conformation of the asubunits so that they can make the appropriateresponse. In addition, a clear distinction can bedrawn between the movements in ligand-bindingdomain and in membrane-spanning domain, wherethe structure is more symmetrical and the subunitscontribute equally to the opening sensitivity ofthe pore.26 The gating appears to occur by fastcooperative movements of helices lining the mem-brane pore, while the ligand-binding domainmay function as a controlling device that eitherdisenables or facilitates these movements.

Ion selectivity and conductance

The three-dimensional structure reveals a scat-tered distribution of charged groups lining theinner walls of both vestibules (Figure 8), and sohighlights the fact that it is the overall effect of manyside-chains that gives rise to the electric fields thatwould influence ion flow through the narrowmembrane pore. This picture complements thefindings of mutagenesis combined with electro-physiological experiments, where the focus isdirected at individual side-chains.Mutagenesis experiments have shown, for

example, that the cation conductance of themuscle-type ACh receptor is strongly diminishedby reduction of the negative charge of the “inter-intermediate” ring (at aE241 in Figure 8(b)) near theintracellular end of M2.21 Also ion selectivity of thea7 ACh receptor was changed from cationic toanionic by altering the charge on this ring, insertinga proline next to it and substituting a residue in thepore.61 Similar changes in ion selectivity have beenobserved with other members of the superfamily onchanging the equivalent amino acid residues nearthe intracellular end of M2.62,63 Consequently, this

Figure 14.Distribution of charged residues onMA helices shaping the intracellular vestibules of transmitter-gated ionchannels: (a) as found in this study for a cation-selective channel; (b) homology model of MA helices of a related anion-selective channel (human glycine receptor a1 subunits; amino acid residues 369–394). The panels show Ca traces, withbars denoting the Ca–Cb bonds of residues having negative (red) or positive charge (blue); the view is from the synapticcleft. In (a) the inside of the vestibule and the windows for the ions are lined predominantly by negatively chargedgroups, whereas in (b) the opposite distribution applies. Also shown on one of the helices in (b) are the locations in the(cation-selective) 5-HT3A receptor of three arginine residues (green bars); the single-channel conductance increasesdramatically when these residues are mutated to neutral or negatively charged residues.65


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local region is sometimes referred to as the chargeselectivity filter of the channel.64 Yet the unper-turbed three-dimensional structure of the AChreceptor shows numerous negatively chargedgroups within the same confined space (Figure8(b)). Evidently, the residues identified by muta-genesis are supplemented by many others to createthe electrostatic environment that is required forefficient cation-selective transport.

The importance of the intracellular vestibule isunderlined by the fact that the equivalent regions inanion-selective members of the superfamily haveopposite distributions of charge. Figure 14 com-pares the MA helices of the ACh receptor with thoseof the (anion-selective) homomeric glycine receptor,after homology alignment. In one case (AChreceptor; Figure 14(a)) the inside of the vestibule islined predominantly by negatively charged groups;in the other (glycine receptor; Figure 14(b)) it islined predominantly by positively charged groups.Thus the whole inner surface of the vestibulecontributes either a cation-stabilising or an anion-stabilising environment, depending on the type oftransmitter-gated channel.

The narrowest region of the membrane pore,determining ion flow through the ACh receptor, isat the intracellular end of M2 near aT244.22 But thelateral windows of the intracellular vestibule areequally, if not more constricting, and in otherchannels may restrict the total flux to the extentthat they play a rate-limiting role. Evidence that thisdoes occur has been obtained recently by combinedmutagenesis and electrophysiological experimentson (cation-selective) 5-HT3 receptors.65 The singlechannel conductance of the recombinant homo-meric 5-HT3A receptor was !1pS, but co-expression with the 5-HT3B subunit increased the

conductance more than 40-fold. The poor conduc-tance of the homomeric channel was attributable tothree arginine residues, which align with thewindow-framing residues of the receptor (greenbars; Figure 14(b)). Mutation of the arginineresidues in the 5-HT3A receptor to their neutral ornegatively charged 5-HT3B counterparts overcamethe anomalously low conductance of the homo-meric channel, as the structure would predict.

Therefore, while the intracellular vestibule of theACh receptor appears to function primarily as anelectrostatic filter, screening out ions of the wrongcharge and size, in other members of the super-family it may have the additional role, related tothe particular subunit combination, of determiningthe conductance of the channel.

General conclusions

This analysis extends earlier electron microscopicanalyses of the ACh receptor in Torpedo post-synaptic membranes, imaged either in the absenceof ACh, or following brief exposure to ACh to trapthe open-channel form. The results together suggestthat the channel has the following properties thatare fundamental to the way it works:

The main ligand-binding a subunits, in theclosed channel, are in a “distorted” state, which isstabilised by inter and intra-subunit interactions.

In the conformational change to open thechannel, the main ligand-binding subunits arethe principal mediators, leading to a concertedrearrangement in the membrane involving all thehelices lining the pore.

The bound ACh opens the channel by causinga localised rearrangement that stabilises the


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alternative “relaxed” conformation of the ligand-binding subunit.

The transition to the open state involvesrotational movements in the ligand-binding sub-units, which unlock their interactions with the pore-lining helices keeping the channel closed.

The gating movements are quite small, beingrestricted energetically by the need to preserve theconserved hydrophobic cores of the subunits.

The ionic surfaces of the vestibules play animportant role in facilitating the selective transportof cations through the channel.

The high level of amino acid sequence conserva-tion, and the functional specificities able to beachieved with chimeric channels,56,66 imply thatall channels of the Cys-loop superfamily areconstructed around the same three-dimensionalframework and function according to the sameglobal principles. These principles applying to theACh receptor are therefore likely to apply, withminor variations, to other members of thesuperfamily.

Methods

Model building

The amino acid sequences of the four T. marmoratapolypeptide chains67,68 were used to create the startingreceptor structure for the refinement. This structure wasmodelled initially by fitting fragments of the chains tothe experimental densities using the program O.32 Themembrane-spanning region was modelled from theoriginal coordinates (PDB entry 1OED). The extracellularregion was modelled from the coordinates of theseparately aligned inner and outer b-sheet fragments ofAChBP,18 omitting at this stage the N-terminal a-helixand most of the connecting loops. The pentagonalstructure shaping the intracellular vestibule was builtfrom stretches of the amino acid sequence that had beententatively identified with this part of the receptor,14 butwhich could not until now be fitted convincingly to thedensities. Further stretches of polypeptide were built intothe model at later stages in the refinement. In the case ofthe extracellular region, these stretches included theN-terminal a-helix, the MIR, the C loop, the Cys loopand the remaining connecting links. In several parts of themap, where the densities could not be interpretedunambiguously, the chains were built into the modelassuming the fold most closely matching that of therelated region in AChBP or in another subunit. Nosignificant densities were found that might correspond toordered lipid molecules or oligosaccharides attached toextracellular portions of the subunits.69

Three-dimensional density map

The 4 A map on which the refinement was conductedhad been derived as a weighted average of the densitiesrepresenting a single receptor, determined from fourhelical families of tube ((K16,6); (K17,5); (K15,7);(K18,6)).10 In each family, the densities were determinedby combining the Fourier terms from the images alongw1500 layer-lines, and imposing the symmetry consistentwith the p2 surface lattice. The criterion for inclusion of

Fourier terms in the data sets was based on signal-to-noise measurement at each point along each layer-line, orPoint Quality (PQ):

PQðR; lÞZffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2FðR; lÞ2cos2ðdfÞ=BKGðR; lÞ2

q; PQðR; lÞR1

where F is the amplitude of the Fourier term at radius Ralong the layer-line l, df is the deviation of its phase fromthe nearest of 08 or 1808, and BKG is the measuredbackground amplitude.33 No additional editing of indi-vidual layer-lines or solvent flattening70 was performed.

Helical structures and phase comparisons

Structures representing each helical family (i.e. havingthe same symmetry elements and average unit celldimensions) were built from the coordinates of themodelled receptor using the program HLXBLD (writtenby M. Stowell). Helical transforms were then calculatedfrom the tube structures to yield the continuous varia-tions in amplitude and phase along layer-lines located atmultiples of the helical repeat. The phase variations alongthe layer-lines, calculated in this way, should ideallymatch the phase variations along the same layer-linesobtained from the Fourier transforms of the images. Inpractice, however, the presence of noise prevents perfectagreement from being achieved.One important source of noise was the overlap of layer-

lines and consequent mixing of Bessel terms havingdifferent orders.14 This gave rise to spurious amplitudesthat were weakened, but not entirely removed by theaveraging. We describe below an objective method usedto weaken the effect of spurious amplitudes by identify-ing and eliminating layer-lines where the amplitudes aredominated by noise. Application of this method broughtabout a significant improvement in the quality of thedensity map, as assessed by comparison between themeasured and calculated phases (Table 1).

Treatment of noise along layer-lines

To distinguish layer-lines containing a weak signalfrom those containing no signal we measured the PQ andF/BKG values averaged over the first (strongest) portionof each layer-line (i.e. from RZn/2prmax to RZn/2prmin,where n is the Bessel order, rmax and rmin are the outer andinner radii of the tube). We then selected layer-lines forretention based on the ratio of the mean values,FmZ �PQ=ð �F= �BKGÞ, i.e. the mean amplitude-weighted“Figure of merit”. It can be shown that Fmz1.414 (i.e.ffiffiffi2

p) for a strong layer-line, and 0.900 (i.e.

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2ð2=pÞ2

p) for a

layer-line containing only random terms. Figure 15 plotsFm versus �F= �BKG for layer-lines of the (K16,6) family.A conservative value of FmZ0.877 for inclusion of thelayer-lines resulted in w20% of them being eliminatedfrom each data set, while the agreement between themeasured and calculated phases in all four casesimproved by 2–4%. We used this threshold figure inderiving the final density map.

Crystallographic refinement

Torsion angle refinement was performed in CNS71

using maximum likelihood (amplitude target), afterplacing the isolated densities corresponding to a singlereceptor in a P1 orthogonal unit cell (Table 2). To facilitatemeasurement of, and correction for possible effects oftube flattening (see Results), the central axis of thereceptor (which lies radially to the axis of the tube) was

Figure 15. Fm plotted against �F=�BKG for individual layer-lines of

the (K16,6) helical family. Thetheoretical figure for Fm, assumingthe layer-line contains only randomterms, is 0.9 (broken line); layer-lines with �F= �BKGO9:0 have notbeen plotted. See the text fordetails.


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aligned with the c axis of the cell and overall anisotropictemperature factors were used. Atomic scattering factorsfor electrons were used without taking account ofchemical bonding effects or charge. The R-factorswere calculated over the range: 100–4 A, with 5% of theterms being used for the calculation of Rfree. Throughoutthe refinement, the value of Rfree was monitored withparallel measurement of the helical phase residuals,obtained by comparing the experimental with calculatedphases from structures of tubes. A reduction in Rfree wasconsidered valid only if it was not accompanied by anincrease in the phase residual. The geometry of the finalmodel was examined with the program PROCHECK.72

The Figures were prepared with the programs MOL-SCRIPT,73 SETOR74 and GRASP.75

Atomic coordinates

The coordinates have been deposited in the ProteinData Bank with accession code 2BG9.

Acknowledgements

The structure refinement was based on data fromelectron images, all of which had been recorded inJapan using microscopes incorporating a liquidhelium-cooled stage. I am particularly grateful toYoshi Fujiyoshi, who designed the stage, and toAtsuoMiyazawa, who recordedmost of the images,for their continued support and advice. The workhas also benefited from many helpful discussionswith colleagues at the Laboratory of MolecularBiology, Cambridge, UK, and at the ScrippsResearch Institute, La Jolla, CA, USA. The researchwas funded, in part, by grants from the European

Commission (QLG3-CT-2001-00902) and NIH(GM61941).

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Edited by W. Baumeister

(Received 20 October 2004; received in revised form 9 December 2004; accepted 15 December 2004)

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