Refined Structure of the Nicotinic Acetylcholine Receptor at 4 Å Resolution

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We present a refined model of the membrane-associated Torpedo acetylcholine (ACh) receptor at 4 Å resolution. An improved experimental density map was obtained from 342 electron images of helical tubes, and the refined structure was derived to an R-factor of 36.7% (Rfree 37.9%) by standard crystallographic methods, after placing the densities corresponding to a single molecule into an artificial unit cell. The agreement between experimental and calculated phases along the helical layer-lines was used to monitor progress in the refinement and to give an independent measure of the accuracy. The atomic model allowed a detailed description of the whole receptor in the closed-channel form, including the ligand-binding and intracellular domains, which have not previously been interpreted at a chemical level. We confirm that the two ligand-binding α subunits have a different extended conformation from the three other subunits in the closed channel, and identify several interactions on both pairs of subunit interfaces, and within the α subunits, which may be responsible for their “distorted” structures. The ACh-coordinating amino acid side-chains of the α subunits are far apart in the closed channel, indicating that a localised rearrangement, involving closure of loops B and C around the bound ACh molecule, occurs upon activation. A comparison of the structure of the α subunit with that of AChBP having ligand present, suggests how the localised rearrangement overcomes the distortions and initiates the rotational movements associated with opening of the channel. Both vestibules of the channel are strongly electronegative, providing a cation-stabilising environment at either entrance of the membrane pore. Access to the pore on the intracellular side is further influenced by narrow lateral windows, which would be expected to screen out electrostatically ions of the wrong charge and size.

Introduction

The nicotinic ACh receptor is a member of the pentameric “Cys-loop” superfamily of transmitter-gated ion channels, which includes neuronal ACh receptors, GABAA receptors, 5-HT3 receptors and glycine receptors.1, 2, 3, 4, 5 The channel is found in high concentrations at the nerve–muscle synapse, where it mediates fast chemical transmission of electrical signals in response to ACh released from the nerve terminal into the synaptic cleft. It is a large (290 kDa) glyco-protein, assembled from a ring of homologous subunits (α, γ, α, β, δ) and divided into three domains: a large N-terminal extracellular ligand-binding domain, a membrane-spanning pore, and a smaller intracellular domain, giving it a total length of about 160 Å normal to the membrane plane. The ligand-binding domain shapes a long, ∼20 Å diameter central vestibule and has two binding sites for ACh, which are about 40 Å from the membrane surface on opposite sides of the pore. The pore makes a narrow water-filled path across the membrane and contains the gate, which opens when ACh occupies both binding sites. The intracellular domain shapes another, smaller vestibule, having narrow lateral openings for the ions.

The receptor subunits in the ligand-binding domain are each organised around two sets of β-sheets packed into a curled β-sandwich and joined through the disulphide bridge forming the Cys loop, as was shown by the structure of the closely related soluble protein, AChBP.6 The ACh-binding sites lie at the α–γ and α–δ subunit interfaces, and are contributed mainly by residues from loops A, B and C, connecting β-strands in the α subunits.7, 8, 9 The subunits in the membrane-spanning domain are each made from four α-helical segments (M1–M4).10 The helical segments are arranged symmetrically, forming an inner ring of helices (M2), which shape the pore, and an outer shell of helices (M1, M3 and M4), which coil around each other and shield the inner ring from the lipids. In the closed channel, the inner ring of helices come together near the middle of the membrane to make a constricting hydrophobic girdle, which constitutes an energetic barrier to ion permeation11, 12 and may function as the gate of the channel.10, 13 The subunits in the intracellular domain each contribute one α-helix (part of the M3–M4 loop), which together make the wall of the vestibule.14

Insight into the structural mechanism of gating has been obtained by electron microscopical experiments on helical tubes grown from Torpedo postsynaptic membranes,15, 16 using a rapid spray-freezing technique to mimic the synaptic release of ACh and trap the open-channel form.17 These experiments showed that binding of ACh initiates two interconnected events in the ligand-binding domain. One is a local disturbance in the region of the ACh-binding sites, and the other a larger-scale conformational change, involving rotational movements predominantly in the two α subunits. The inner M2 helices also change their configuration in response to ACh, widening the lumen of the pore at the middle of the membrane. Higher resolution studies of the extended conformational change18 and of the structure in the membrane10 suggested a simplified mechanical model for the channel opening mechanism, whereby ACh triggers rotations of the inner β-sheets of the α subunits and the twisting movement, communicated through the inner helices, breaks the gate apart.

In addition to the structural details, summarised above, the roles played by individual amino acid residues in determining the ligand-binding, gating and cation-conduction properties of the ACh receptor have been extensively characterised by chemical labelling and by site-directed mutagenesis experiments combined with electrophysiological study of function.19, 20, 21, 22, 23, 24, 25, 26, 27, 28 Other experiments of this kind, performed on GABAA, glycine, 5-HT3 and neuronal α7 receptors constitute a wealth of complementary information.

We report here a preliminary three-dimensional framework for relating these biochemical and physiological data, based on refinement of a 4 Å structure obtained from electron images of the tubular Torpedo membranes frozen in a near-physiological ionic environment.10 The refined model enables a detailed description of the whole receptor in the closed-channel form, including the ligand-binding region and vestibular entrances, which have not previously been interpreted at a chemical level. We confirm that the two ligand-binding α subunits have a different extended conformation from the three other subunits in the closed-channel form of the receptor,18 and identify several interactions at the subunit interfaces, and within the α subunits, which may be responsible for their “distorted” structures. The ACh-binding site itself, which was not correctly identified in the 9 Å map,13 shows many features that are apparent in the structure of AChBP. However, the organisation of the B and C loops at the binding site of the closed channel differs from that in AChBP, where ligand is present, indicating that the binding reaction is accompanied by a local structural rearrangement. A comparison of the two structures suggests how the local rearrangement associated with ACh binding stabilises the alternative open-channel form of the receptor. Given our improved understanding of this initial step, it is now possible to sketch a complete picture of the series of coordinated events leading to opening of the channel. Finally, we discuss the role of the vestibules, the ionic surfaces of which create a strongly electronegative environment at either entrance of the narrow membrane pore.

Section snippets

Structure refinement

The original 4 Å data set was from 359 images of tubes,10 grown from Torpedo marmorata postsynaptic membranes.15 The tubes have four distinct helical symmetries, with individual molecules arranged on a p2 surface lattice15 such that the inside of the tube corresponds to the inside of the cell.29 The receptors come closest to each other near radial 2-fold axes (Figure 1(a)). A disulphide bridge between the δ subunits of neighbouring receptors30, 31 lies near the membrane at one such axis; the

Symmetry

The approximate 5-fold symmetry of the receptor was examined further by determining the angles required to achieve optimal least-squares superposition of the subunits around the pentamer. Deviations from 5-fold were found to be smallest in the membrane-spanning domain, where each subunit assumed an orientation lying within 2° (s.d.=1.61°) of the value required for exact register with a 5-fold-averaged structure. These deviations appeared to be a consequence of structural variations (which are

Discussion

The refined 4 Å structure reported here provides a chemical interpretation of all the main functional regions of the ACh receptor, as they would appear under near-physiological ionic conditions in Torpedo postsynaptic membranes. Although the final crystallographic R-factor was only 36.7%, limited by the quality of the amplitudes from images, we demonstrated that the polypeptide chains could now be traced with reasonable confidence over the entire length of the molecule (Figure 2, Figure 12).

General conclusions

This analysis extends earlier electron microscopic analyses of the ACh receptor in Torpedo postsynaptic membranes, imaged either in the absence of ACh, or following brief exposure to ACh to trap the open-channel form. The results together suggest that the channel has the following properties that are fundamental to the way it works:

The main ligand-binding α subunits, in the closed channel, are in a “distorted” state, which is stabilised by inter and intra-subunit interactions.

In the

Model building

The amino acid sequences of the four T. marmorata polypeptide chains67, 68 were used to create the starting receptor structure for the refinement. This structure was modelled initially by fitting fragments of the chains to the experimental densities using the program O.32 The membrane-spanning region was modelled from the original coordinates (PDB entry 1OED). The extracellular region was modelled from the coordinates of the separately aligned inner and outer β-sheet fragments of AChBP,18

Acknowledgements

The structure refinement was based on data from electron images, all of which had been recorded in Japan using microscopes incorporating a liquid helium-cooled stage. I am particularly grateful to Yoshi Fujiyoshi, who designed the stage, and to Atsuo Miyazawa, who recorded most of the images, for their continued support and advice. The work has also benefited from many helpful discussions with colleagues at the Laboratory of Molecular Biology, Cambridge, UK, and at the Scripps Research

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