Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.     

Core 1 glycans on alpha-dystroglycan mediate laminin-induced acetylcholine receptor clustering but not laminin binding

Although unique O-linked oligosaccharides on alpha-dystroglycan are important for binding to a variety of extracellular ligands, the function(s) of more generic carbohydrate structures on alpha-dystroglycan remain unclear. Recent studies suggest a role for glycoconjugates bearing the core 1 disaccharide Galbeta(1-3)GalNAc in acetylcholine receptor (AChR) clustering on the surface of muscle cells. Here, we report experiments demonstrating that the core 1-specific lectin jacalin almost completely abrogated laminin-induced AChR clustering in C2C12 myotubes and that alpha-dystroglycan was the predominant jacalin-binding protein detected in C2C12 myotube lysates. Although jacalin likely inhibited laminin-induced AChR clustering by directly binding to alpha-dystroglycan, jacalin had no effect on laminin binding to the myotube surface or to alpha-dystroglycan. Like jacalin, peanut agglutinin lectin also binds the core 1 disaccharide but not when it is terminally sialylated as expressed on alpha-dystroglycan. We show that C2C12 alpha-dystroglycan bound to peanut agglutinin only after digestion with neuraminidase. Simultaneous treatment of myotubes with neuraminidase and endo-O-glycosidase diminished alpha-dystroglycan binding to peanut agglutinin and inhibited neuraminidase-induced AChR clustering. We conclude that sialylated core 1 oligosaccharides of alpha-dystroglycan are important for laminin-induced AChR clustering and that their function in this process is distinct from the established role of alpha-dystroglycan oligosaccharides in laminin binding.     

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.     

Tryptophan-scanning mutagenesis in the alphaM3 transmembrane domain of the muscle-type acetylcholine receptor. A spring model revealed

Membrane proteins constitute a large fraction of all proteins, yet very little is known about their structure and conformational transitions. A fundamental question that remains obscure is how protein domains that are in direct contact with the membrane lipids move during the conformational change of the membrane protein. Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. Here, we use the tryptophan-scanning mutagenesis to monitor the conformational change of the alphaM3 domain of the muscle-type AChR. The perturbation produced by the systematic tryptophan substitution along the alphaM3 domain were characterized through two-electrode voltage clamp and 125I-labeled alpha-bungarotoxin binding. The periodicity profiles of the changes in AChR expression (closed state) and ACh EC50 (open-channel state) disclose two different helical structures; a thinner-elongated helix for the closed state and a thicker-shrunken helix for the open-channel state. The existence of two different helical structures suggest that the conformational transition of the alphaM3 domain between both states resembles a spring motion and reveals that the lipid-AChR interface plays a key role in the propagation of the conformational wave evoked by agonist binding. In addition, the present study also provides evidence about functional and structural differences between the alphaM3 domains of the Torpedo and muscle-type receptors AChR.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.     

t-PA K1区的同源模建及Kringle区与Lysine相互作用的研究

利用同源模建方法预测了ｔ－ＰＡＫ１区的三维结构。通过结构叠合确定了ｔ－ＰＡＫ１、Ｋ２区,纤溶酶原Ｋ１、Ｋ４区及ＵＫ Ｋ区的Ｌｙｓｉｎｅ结合口袋。静电势计算及疏水性分析表明,在ｔ－ＰＡ Ｋ２Ｃ Ａ２区以及纤溶酶原Ｋ１、Ｋ４区与纤维蛋白裸露的Ｌｙｓｉｎｅ之间在明显的的静电势互补和疏水面契合。确定了Ｋｒｉｎｇｌｅ区结合口袋与Ｌｙｓｉｎｅ亲和重要所基酸,分析了ｔ－ＰＡＫ１区,ＵＫ Ｋ区不能结合Ｌｙｓｉｎｅ     

The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.     

Differential scanning calorimetry and Fourier transform infrared analysis of lipid-protein interactions involving the nicotinic acetylcholine receptor

Lipid-protein interactions were studied using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into dielaidoylphosphatidylcholine (DEPC, 18:1 trans-9,10) membranes. The structural and thermodynamic behavior of lipids in the vicinity of the protein were studied by differential scanning calorimetry and Fourier transform infrared spectroscopy. The effects of AChR on the thermodynamic parameters associated with lipid phase transitions were to reduce the enthalpy change, lower the transition temperature and reduce the cooperative behavior of the lipid molecules. A stoichiometry of approx. 95 lipids per AChR molecule was found by simulating the decrease in enthalpy in terms of a simple model in which a fixed number of lipid molecules are prevented from undergoing a cooperative phase transition. In parallel, the vibrational spectra of pure DEPC and AChR reconstituted in DEPC membranes at various lipid to protein ratios were examined. Profiles of the 3000-2800 cm-1 C-H stretching region and 1350-950 cm-1 characteristic of the headgroup region of the lipid exhibit little sensitivity to protein/lipid ratio reflecting weak interaction of AChR with DEPC. The lipid carbonyl on the other hand appear to be increasingly hydrogen bonded in the presence of AChR. The results provide new information about the size and physical state of the motionally restricted lipid environment that surrounds the acetylcholine receptor. The results are discussed in the context of lipid-mediated alterations in acetylcholine receptor function.     

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind alpha-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the alpha-BTX/AChR system yielded 13-mer peptides that bind to alpha-BTX with high affinity and are potent inhibitors of alpha-BTX binding to AChR (IC(50) of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel beta-hairpin structure when bound to alpha-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.     

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.     

Thymopoietins and long postsynaptic neurotoxins share common information in their primary structures

The informational content of the primary structure of thymopoietin (TP) is investigated using the informational spectrum method (ISM). We show that the sequence of TP shares common information with the sequences of long postsynaptic snake neurotoxins, although no apparent similarity was found among their primary structures. The most sensitive point in the sequence of TP, concerning this information, is D-34, previously determined as being the residue responsible for TP's effect on neuromuscular transmission. Our results suggest that TP and long toxins recognize the neuromuscular nicotinic acetylcholine receptor (AChR) and/or bind to the AChR in a different mode than the short toxins do.     

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.     

Fine localization of the major alpha-bungarotoxin binding site to residues alpha 189-195 of the Torpedo acetylcholine receptor. Residues 189, 190, and 195 are indispensable for binding

alpha-Bungarotoxin blocks acetylcholine-mediated ion channel opening of peripheral acetylcholine receptors (AChR). A major binding region for alpha-bungarotoxin has been recently identified within parts of the segment 170-204 of the alpha-subunit. We used the Pepscan systematic peptide synthesis system to determine the minimum Torpedo AChR segment required for alpha-bungarotoxin binding and to investigate the role of each residue within this segment. Continuously overlapping decapeptides within alpha 179-203 and several decapeptides covering other alpha-subunit sequences showed that alpha 188-197 and alpha 189-198 exhibited the best 125I-alpha-bungarotoxin binding activity (KD = 7.3 x 10(-8) and 4.3 x 10(-8) M, respectively). Several continuously overlapping nona-, octa-, hepta-, hexa-, and tetrapeptides showed that the heptapeptide alpha 189-195 was the minimum sequence with high binding activity (KD = 5.6 x 10(-8)M). d-Tubocurarine, but not carbamylcholine, blocked toxin binding. Twenty-six analogs of the alpha 188-197, most having 1 residue substituted by Ala or Gly, showed that Tyr189, Tyr190, and especially Asp195 were indispensable for 125I-alpha-bungarotoxin binding. Cys192 and Cys193 could be substituted by other amino acids, proving that the disulfide bond between alpha 192-193 was not required for alpha-bungarotoxin binding. The decreased alpha-bungarotoxin binding capacity of the equivalent human muscle AChR alpha 188-197 peptide was the result of substitution of Tyr by Thr at alpha 189.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

The sites of neurotoxicity in alpha-cobratoxin

We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the acetylcholine receptor protein from Torpedo marmorata. (i) The toxin derivatives which were fully modified at lysine (penta-epsilon-N,N-dimethyl lysine; penta-epsilon-N-acetyl lysine), arginine (penta-N7,N8-(1,2-dihydroxycyclohex-1,2-ylene arginine), and tyrosine (mononitrotyrosine) all had significant remaining toxicity and affinity of binding. (ii) The "extra" disulfide of alpha-cobratoxin was selectively reduced and alkylated. Depending on the charge, size, and hydrophobicity of the attached groups, derivatives were obtained that bound to the acetylcholine receptor with higher (di-S-carboxyamidomethyl), about equal (di-S-pyridylethyl), or lower (di-iodoacetaminoethylnaphthylamine-5-sulfonic acid) affinity than the unmodified toxin. (iii) A fully reduced and carbamidomethylated derivative of alpha-cobratoxin obtained by repeating the procedure for selective reduction six times still bound with appreciable affinity (KD approximately 3 X 10(-6) M) to the acetylcholine receptor. We conclude that neither a single positively charged residue nor tyrosine nor the integrity of the disulfides is absolutely essential for toxicity. Furthermore, the single tyrosine and the area around the extra disulfide do not participate in the binding to the receptor. Together with previous findings on this interaction, this suggests a multipoint attachment of toxin and receptor involving several locally separate structural elements of the toxin.     

Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding

The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. halpha-(1-210) bound (125)I-alpha-bungarotoxin with a high affinity (K(d) = 5.1 +/- 2.4 nm), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K(i) approximately 7.5 mm). Interestingly, (125)I-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to halpha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alpha subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i