Inhibition mechanism of the acetylcholine receptor by alpha-neurotoxins as revealed by normal-mode dynamics

The nicotinic acetylcholine receptor (AChR) is the prototype of ligand-gated ion channels. Here, we calculate the dynamics of the muscle AChR using normal modes. The calculations reveal a twist-like gating motion responsible for channel opening. The ion channel diameter is shown to increase with this twist motion. Strikingly, the twist motion and the increase in channel diameter are not observed for the AChR in complex with two alpha-bungarotoxin (alphaBTX) molecules. The toxins seems to lock together neighboring receptor subunits, thereby inhibiting channel opening. Interestingly, one alphaBTX molecule suffices to prevent the twist motion. These results shed light on the gating mechanism of the AChR and present a complementary inhibition mechanism by snake-venom-derived alpha-neurotoxins.     

NMR structure of alpha-bungarotoxin free and bound to a mimotope of the nicotinic acetylcholine receptor

A combinatorial library approach was used to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. Among the sequences, which inhibited binding of alpha-bungarotoxin to muscle and neuronal nicotinic receptors, HRYYESSLPWYPD, a 14-amino acid peptide with considerably higher toxin-binding affinity than the other synthesized peptides, was selected, and the structure of its complex with the toxin was analyzed by NMR. Comparison of the solution structure of the free toxin and its complex with this peptide indicated that complex formation induced extensive conformational rearrangements mainly at finger II and the carboxy terminus of the protein. The peptidyl residues P10 and Y4 seemed to be critical for peptide folding and complex stability, respectively. The latter residue of the peptide strongly interacted with the protein by entering a small pocket delimited by D30, C33, S34, R36, and V39 toxin side chains.     

Electrostatic resistance to alpha-neurotoxins conferred by charge reversal mutations in nicotinic acetylcholine receptors

The evolution of venom resistance through coevolutionary chemical arms races has arisen multiple times throughout animalia. Prior documentation of resistance to snake venom α-neurotoxins consists of the N-glycosylation motif or the hypothesized introduction of arginine at positions 187 at the α-1 nicotinic acetylcholine receptor orthosteric site. However, no further studies have investigated the possibility of other potential forms of resistance. Using a biolayer interferometry assay, we first confirm that the previously hypothesized resistance conferred by arginine at position 187 in the honey badger does reduce binding to α-neurotoxins, which has never been functionally tested. We further discovered a novel form of α-neurotoxin resistance conferred by charge reversal mutations, whereby a negatively charged amino acid is replaced by the positively charged amino acid lysine. As venom α-neurotoxins have evolved strong positive charges on their surface to facilitate binding to the negatively charged α-1 orthosteric site, these mutations result in a positive charge/positive charge interaction electrostatically repelling the α-neurotoxins. Such a novel mechanism for resistance has gone completely undiscovered, yet this form of resistance has convergently evolved at least 10 times within snakes. These coevolutionary innovations seem to have arisen through convergent phenotypes to ultimately evolve a similar biophysical mechanism of resistance across snakes.     

Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.     

NMR mapping and secondary structure determination of the major acetylcholine receptor alpha-subunit determinant interacting with alpha-bungarotoxin

The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Mapping by synthetic peptides of the binding sites for acetylcholine receptor on alpha-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of -bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).     

Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.     

The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.     

Benzophenone-type photoactivatable derivatives of alpha-neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.     

Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and mu-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.     

Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides

Antibodies were raised to the amino- and car?y-terminal decapeptides of Torpedo californica acetylcholine receptor. Structural studies of the native receptor using the antipeptide antibodies as probes proved the existence of the car?y terminal sequence in the α subunit predicted from its cDNA sequence and supported structural models of the native receptor that place the car?y termini on the intracellular side. The amino termini of the subunits were not accessible on the surface of native receptor.     

Analysis of receptor-ligand interactions using nitrocellulose gel transfer: application to Torpedo acetylcholine receptor and alpha-bungarotoxin

It has been shown that the retention times of oligosaccharides containing N-acetyl amino sugars in reverse-phase high-performance liquid chromatography are sensitive both to chain length and to stereochemical differences. Data showing that oligosaccharides of human milk can be fractionated according to stereochemistry on C-18 columns using water as eluant, is presented. Detection at the nanomolar level is possible using ultraviolet absorption at 190 nm as a result of the acetamido group. The separation of linkage isomers by fractionation of two tetrasaccharides of identical carbohydrate composition, lacto-N-tetraose and lacto-N-neotetraose, and by the separation of two isomeric pentasaccharides, lacto-N-fucopentaose I and II, is shown. In some cases it was possible to preparatively fractionate pure oligosaccharides from complex mixtures. The identity and purity of the oligosaccharides was determined by proton-nuclear magnetic resonances spectroscopy at 300 MHz using the method of Vliegenthart and co-workers (B. Fournet, G. Montreuil, L. Strecker, J. Dorland, J. Haverkamp, J. F. G. Vliegenthart, J. P. Binette, and K. Schmid (1978) Biochemistry 17(24), 5206; J. F. G. Vliegenthart, H. V. Halbeek, and L. Dorland (1981) Pure Appl. Chem. 53, 45) in which "structural reporter" protons are identified for the different isomeric oligosaccharides. In addition to assignment of the anomeric proton resonances, it was possible to assign H4 of galactose linked at O3 as well as fucose H5 and H6 resonances.     

The mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin, a protein isolated from the synapse-rich electric organ of Torpedo californica, induces the formation of specializations on myotubes in culture which resemble the post-synaptic apparatus at the vertebrate skeletal neuromuscular junction. For example, the specializations contain aggregates of acetylcholine receptors and acetylcholinesterase. This report summarizes the evidence that the formation of the post-synaptic apparatus at developing and regenerating neuromuscular junctions is triggered by the release of agrin from motor axon terminals and describes results of recent experiments which suggest that agrin-induced tyrosine phosphorylation of the beta subunit of the acetylcholine receptor may play a role in receptor aggregation.     

Sensitivity to voltage-independent inhibition determined by pore-lining region of the acetylcholine receptor.

Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). This study characterizes the mechanism of selective long-term inhibition of neuronal and muscle-neuronal chimeric nAChRs by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (bis-TMP-10 or BTMPS), a bifunctional form of the potent ganglionic blocker tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been previously demonstrated to arise, at least in part, from the binding of the bis compound to neuronal beta-subunits. In this study, long-term inhibition is demonstrated to be dependent upon the presence of sequence element(s) within the pore-lining second transmembrane domain (tm2) of neuronal beta-subunits; however, the inhibitor binding site itself does not appear to be contained within the segment of the channel pore influenced by the membrane electric field. Specifically, our results imply that bis-TMP-10 interacts with an activation-sensitive element, the availability of which may be regulated by a sequence in the tm2 domain. Furthermore, we demonstrate a compound length requirement for long-term inhibition that would be consistent with binding to multiple sites located on the extracellular portion of the receptor.     

Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor

We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).     

Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France     

A modification of alpha-bungarotoxin amino group residues with retention of binding properties to isolated and membrane-bound acetylcholine receptor

α-Bungarotoxin (α-Bgt), an α-neurotoxin, has been ¹⁴C-methylated by treatment with [¹⁴C]formaldehyde following NaCNBH₃ reduction. The methylation rate is fast (about 84% methylation in 15 min), with 12 methyl groups incorporated per mole of α-Bgt or a mean of 1.7 methyl groups per available amine residue. The specific activity of α-[¹⁴C]Bgt is 768 mCi/mmol. Unlike most of the reported chemical modifications of α-neurotoxins, involving a high decrease of the toxin activity after modification, α-[¹⁴C]Bgt retains 100% of its unmodified ability to bind to both isolated acetylcholine receptor (AcChR) and AcChR-enriched membrane fragments prepared from Torpedo californica. This lysyl residue modification does not perturb the toxin binding activity, probably, because the net positive charges of the ?-amino groups and amino-terminal residue remain unaltered. ¹⁴C-Methylated α-Bgt appears better suited than ¹²⁵I-α-Bgt for use in AcChR binding studies because of the longer half-life of the isotope, and the apparent high uniformity of labeling of the toxin preparations.