Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors

Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.     

Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor

Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.     

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.     

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.     

Specificity of the basic side chains of Lys114, Lys125, and Arg129 of antithrombin in heparin binding

The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high-affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor.     

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding alpha-bungarotoxin

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

Roles of N-terminal region residues Lys11, Arg13, and Arg24 of antithrombin in heparin recognition and in promotion and stabilization of the heparin-induced conformational change

The N-terminal region residues, Lys11, Arg13, and Arg24, of the plasma coagulation inhibitor, antithrombin, have been implicated in binding of the anticoagulant polysaccharide, heparin, from the identification of natural mutants with impaired heparin binding or by the X-ray structure of a complex of the inhibitor with a high-affinity heparin pentasaccharide. Mutations of Lys11 or Arg24 to Ala in this work each reduced the affinity for the pentasaccharide approximately 40-fold, whereas mutation of Arg13 to Ala led to a decrease of only approximately 7-fold. All three substitutions resulted in the loss of one ionic interaction with the pentasaccharide and those of Lys11 or Arg24 also in 3-5-fold losses in affinity of nonionic interactions. Only the mutation of Lys11 affected the initial, weak interaction step of pentasaccharide binding, decreasing the affinity of this step approximately 2-fold. The mutations of Lys11 and Arg13 moderately, 2-7-fold, altered both rate constants of the second, conformational change step, whereas the substitution of Arg24 appreciably, approximately 25-fold, reduced the reverse rate constant of this step. The N-terminal region of antithrombin is thus critical for high-affinity heparin binding, Lys11 and Arg24 being responsible for maintaining appreciable and comparable binding energy, whereas Arg13 is less important. Lys11 is the only one of the three residues that is involved in the initial recognition step, whereas all three residues participate in the conformational change step. Lys11 and Arg13 presumably bind directly to the heparin pentasaccharide by ionic, and in the case of Lys11, also nonionic interactions. However, the role of Arg24 most likely is indirect, to stabilize the heparin-induced P-helix by interacting intramolecularly with Glu113 and Asp117, thereby positioning the crucial Lys114 residue for optimal ionic and nonionic interactions with the pentasaccharide. Together, these findings show that N-terminal residues of antithrombin make markedly different contributions to the energetics and dynamics of binding of the pentasaccharide ligand to the native and activated conformational states of the inhibitor that could not have been predicted from the X-ray structure.     

Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit.

To facilitate structural studies of the ligand binding region from the nicotinic acetylcholine receptor (nAChR), we have developed methods for the high-level expression and purification of an important functional portion of the N-terminal extracellular domain (ECD) of the alpha-subunit. Two soluble receptor fragments comprising residues 143-210 of the Torpedo californica alpha-subunit were expressed in E. coli: alphaT68His6, which contains a histidine tag, and alphaT68M1, which includes the first transmembrane region, M1, of the alpha-subunit. Both proteins demonstrate saturable, high-affinity alpha-bungarotoxin (Bgtx) binding with an apparent equilibrium KD (3 nM) that is comparable to the affinities reported for preparations comprising the entire alpha-subunit ECD. These results demonstrate that the ECD determinants required for Bgtx recognition of the alpha-subunit are entirely specified by residues 143-210. The binding of small ligands was demonstrated in competition assays with 125I-Bgtx yielding KI values of 58 and 105 microM for d-tubocurarine and nicotine, respectively. Circular dichroism (CD) analysis of monomeric alphaT68His6 protein revealed considerable secondary structure. Furthermore, a cooperative, two-state folding transition was observed upon urea denaturation. To circumvent concentration-dependent aggregation of the alphaT68His6 protein at the millimolar concentrations needed for NMR study, we utilized the M1 transmembrane domain to anchor the recombinant receptor fragment onto membrane-mimicking micelles. Monodispersed preparations of alphaT68M1 in dodecylphosphocholine micelles demonstrate high-affinity Bgtx binding and considerable secondary structure by CD. The structural features revealed in the CD profile appear to undergo a cooperative, two-state folding transition upon thermal denaturation. Initial NMR studies suggest that micellar preparations of the alphaT68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.     

Structural determinants within residues 180-199 of the rodent alpha 5 nicotinic acetylcholine receptor subunit involved in alpha-bungarotoxin binding

Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) alpha subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. We have previously defined alpha-bungarotoxin (alpha-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR alpha subunit, designated alpha 5 [McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 9816-9824], and between residues 181 and 200 of the chick neuronal alpha 7 and alpha 8 subunits [McLane, K. E., Wu, X., Schoepfer, R., Lindstrom, J., & Conti-Tronconi, B. M. (1991) J. Biol. Chem. (in press)]. These sequences are relatively divergent compared with the Torpedo and muscle nAChR alpha 1 alpha-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the alpha 5 sequence, we were interested in determining the critical amino acid residues for alpha-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat alpha 5(180-199) sequence were tested, using a competition assay, in which peptides compete for 125I-alpha-BTX binding with native Torpedo nAChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.     

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.     

Substitution of Torpedo acetylcholine receptor alpha 1-subunit residues with snake alpha 1- and rat nerve alpha 3-subunit residues in recombinant fusion proteins: effect on alpha-bungarotoxin binding.

A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector. Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind alpha-bungarotoxin. The fusion protein of the Torpedo sequence bound 125I-alpha-bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data). Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding. Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced alpha-bungarotoxin binding to undetectable levels. The P194L mutation alone abolished toxin binding. Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable alpha-bungarotoxin binding. Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding. It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of alpha-bungarotoxin binding. Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.     

Binding of a curarimimetic toxin from cobra venom to the nicotinic acetylcholine receptor. Interactions of six biotinyltoxin derivatives with receptor and avidin

We have reacted N-hydroxysuccinimidyl biotin with the principal curarimimetic toxin in Naja naja siamensis venom, biotinylating each of the five lysine residues and the N-terminal isoleucine. The six monobiotinyl-toxins were isolated by ion-exchange chromatography, and the residue modified in each was identified by peptide mapping and amino acid analysis. We evaluated the role of each lysine in the binding of toxin to the acetylcholine receptor by measuring the affinity of each biotinyltoxin for receptor and by determining which biotinyltoxins could bind receptor and avidin simultaneously. The effect of biotinylation of each residue decreased the affinity of toxin for receptor in the order Lys 23 greater than Lys 49 greater than Lys 35 greater than Lys 69 congruent to Lys 12 greater than Ile 1. Biotinyltoxin modified either at Lys 12 or at Lys 69 is effective in cross-linking avidin to receptor, while biotinyltoxin modified at Lys 49 can form a low-affinity avidin-biotinyltoxin-receptor complex. Taken together, these results help define the surface of toxin that binds to receptor.     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding

Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.     

The effect of chemical modification on the immunochemical activity of Rm-III neurotoxin from the anemone Radiantus macrodactylus]

A chemical modification was used for studying the organization of antigenic determinants of neurotoxin Rm-III from the sea anemone Radianthus macrodactylus. Immunochemical experiments were performed using competitive enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies to Rm-III. The modification affected N-terminal amino group of Gly1, Lys4, Arg13, Trp30 residues, a residue in the Lys46-Lys47-Lys48 sequence, two different residues in the Asp6-Asp7-Glu8 sequence in two samples of the toxin, and two disulphide bonds. Only the modification of the disulphide bonds led to a considerable change in the toxin's affinity to antibodies. The modification of Trp30 resulted in two-fold decrease of the toxin concentration necessary for 50% inhibition of the test-system, whereas upon modification of any other amino acid residue this concentration increased but not more than by 2.2 times. It is suggested that Rm-III sequence lacks individual residues which are of great importance for the toxin's antigenic activity, its conformation being of vital importance for the formation of the toxin's antigenic determinants.