Do structural deviations between toxins adopting the same fold reflect functional differences?

Three-finger proteins form a structurally related family of compounds that exhibit a great variety of biological properties. To address the question of the prediction of functional areas on their surfaces, we tentatively conferred the acetylcholinesterase inhibitory activity of fasciculins on a short-chain curaremimetic toxin. For this purpose, we assimilated the three-dimensional structure of fasciculin 2 with the one of toxin alpha. This comparison revealed that the tips of the first and second loops, together with the C terminus residue, deviated most. A first recombinant fasciculin/toxin alpha chimera was designed by transferring loop 1 in its entirety together with the tip of loop 2 of fasciculin 2 into the toxin alpha scaffold. A second chimera (rChII) was obtained by adding the point Asn-61 --> Tyr substitution. Comparison of functional and structural properties of both chimeras show that rChII can accommodate the imposed modifications and displays nearly all the acetylcholinesterase-blocking activities of fasciculins. The three-dimensional structure of rChII demonstrates that rChII adopts a typical three-fingered fold with structural features of both parent toxins. Taken together, these results emphasize the great structural flexibility and functional adaptability of that fold and confirm that structural deviations between fasciculins and short-chain neurotoxins do indeed reflect functional diversity.     

Stability of a structural scaffold upon activity transfer: X-ray structure of a three fingers chimeric protein

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones. Loop I, half of loop II and the C-terminal residue of fasciculin 2 were therefore transferred into the toxin alpha. The inhibition constant of the resulting chimera is only 15-fold lower than that of fasciculin 2, and as expected the potency of binding to the toxin alpha target has been lost. In order to understand the structure-function relationship between the chimera and its "parent" molecules, we solved its structure by X-ray crystallography. The protein crystallized in space group P3(1)21 with a=b=58.5 A, and c=62.3 A. The crystal structure was solved by molecular replacement and refined to 2.1 A resolution. The structure belongs to the three-fingered snake toxin family with a core of four disulphide bridges from which emerge the three loops I, II and III. Superimposition of the chimera on fasciculin 2 or toxin alpha revealed an overall fold intermediate between those of the two parent molecules. The regions corresponding to toxin alpha and to fasciculin 2 retained their respective geometries. In addition, the chimera protein displayed a structural behaviour similar to that of fasciculin 2, i.e. dimerization in the crystal structure of fasciculin 2, and the geometry of the region that binds to acetylcholinesterase. In conclusion, this structure shows that the chimera retains the general structural characteristics of three-fingered toxins, and the structural specificity of the transferred function.     

The fasciculin-acetylcholinesterase interaction

Acetylcholinesterase (AChE) terminates the action of the neurotransmitter acetylcholine at cholinergic synapses in the central and peripheral nervous systems. Fasciculins, which belong to the family of "three-fingered" snake toxins, selectively inhibit mammalian AChEs with Ki values in the picomolar range. In solution, the cationic fasciculin appears to bind to the enzyme's peripheral anionic site, located near the mouth of the gorge leading to the active center, to inhibit catalysis either allosterically or by creating an electrostatic barrier at the gorge entry (or both). Yet the crystal structure of the fasciculin-mouse AChE complex, which shows that the central loop of fasciculin fits snugly at the entrance of the gorge, suggests that the mode of action of fasciculin is steric occlusion of substrate access to the active center. Mutagenesis of the fasciculin molecule, undertaken to establish a functional map of the binding surfaces, identified determinants common to those identified by the structural approach and revealed that only a few of the many fasciculin residues residing at the complex interface provide the strong contacts required for high affinity binding and enzyme inhibition. However, it did not reconcile the disparity between the kinetic and structural data. Finally, the crystal structure of mouse AChE without bound fasciculin shows a tetrameric assembly of subunits; within the tetramer, a short loop at the surface of a subunit associates with the peripheral site of a facing subunit and sterically occludes the entrance of the active center gorge. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of fasciculin bound to AChE. This suggests not only that the peripheral site of AChE is a site for association of heterologous proteins with interactive surface loops, but also that endogenous peptidic ligands of AChE sharing structural features with the fasciculin molecule might exist.     

Fasciculins, anticholinesterase toxins from the venom of the green mamba Dendroaspis angusticeps

Two toxins that are potent inhibitors of acetylcholinesterase have been isolated from the venom of the green mamba, Dendroaspis angusticeps. The toxins have been called fasciculins since after injection into mice (i.p. 0.5-3 micrograms/g body weight) they cause severe, generalized and long-lasting (5-7 h) fasciculations. Homogenates of diaphragm, tibialis anterior and gastrocnemius muscles from mice injected with fasciculins showed a decrease in acetylcholinesterase activity by 45-60% compared to muscles from control animals. Histochemical staining revealed a greatly reduced acetylcholinesterase activity at neuromuscular junctions. Fasciculins have 61 amino acid residues and four disulfides. The molecular weights are 6765 (fasciculin 1) and 6735 (fasciculin 2). The sequences of the two toxins differ probably only at one position by a replacement of Tyr with Asp/Asn. 1 g of venom contained about 40 mg of fasciculins, 2/3 of which was fasciculin 2. A similar inhibitor has also been isolated from D. polylepis (black mamba) venom. The sequence of fasciculin 2 is known. Most of the positive charges are concentrated in a small section of the central part of the molecule, and most of the negative charges are in the C-terminal region. Fasciculins appear to have a pronounced dipole character. Fasciculin binds to the peripheral anionic site, since it can displace propidium, a probe for that site, from acetylcholinesterase. In vitro, in Krebs-Henseleit solution containing 2 mM NaH2PO4 (pH 7.4), fasciculin 2 inhibits acetylcholinesterase from human erythrocytes (Ki = 1.1 X 10(-10) M, 37 degrees C), rat muscle (Ki = 1.2 X 10(-10) M, 37 degrees C) and Electrophorus electricus (Ki = 3.0 X 10(-10) M, 22 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

Fasciculin II,a protein inhibitor of acetylcholinesterase,tested on central synapses ofAplysia

Fasciculin II, a potential inhibitor of acetylcholinesterase (AChE), was tested on two types of Aplysia cholinergic receptors: H type, opening Cl- channels; and D type, opening cationic channels. Evoked postsynaptic inhibitory responses and responses to ionophoretic application of acetylcholine (ACh) or carbachol onto H-type receptors were potentiated in the presence of fasciculin II at 10(-9) M, whereas the same concentration of this drug was without effect on the evoked postsynaptic excitatory responses or on the application of ACh or carbachol on D-type receptors. The observed effects of fasciculin II were identical to those obtained with other inhibitors of AChE on the same preparation. The facilitatory effect on the carbachol response in H-type cells indicates that fasciculin II, as other AChE inhibitors, does not act on H-type synapses solely by blocking the hydrolysis of ACh. We concluded that fasciculin II was a good inhibitor of acetylcholinesterase on neuronal preparations in vivo. The results are further discussed as a new element in favor of a previously proposed hypothesis of a molecular interaction between AChE and ACh H-type receptors.     

Modification of substrate inhibition of synaptosomal acetylcholinesterase by cardiotoxins

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.     

Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cysteines

To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k(cat)/K(m)) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.     

Synthesis and characterization of a chimeric peptide derived from fasciculin that inhibits acetylcholinesterase.

Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.     

Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.     

Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.     

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.     

Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.     

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5? resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.     

Computational studies of acetylcholinesterase complexed with fullerene derivatives: a new insight for Alzheimer disease treatment

Here, we propose five fullerene (C60) derivatives as new drugs against Alzheimer’s disease (AD). These compounds were designed to act as new human acetylcholinesterase (HssAChE) inhibitors by blocking its fasciculin II (FASII) binding site. Docking and molecular dynamic results show that our proposals bind to the HssAChE tunnel entrance, forming stable complex, and further binding free energy calculations suggest that three of the derivatives proposed here could be potent HssAChE inhibitors. We found a region formed by a set of residues (Tyr72, Asp74, Trp286, Gln291, Tyr341, and Pro344) which can be further exploited in the drug design of new inhibitors of HssAChE based on C60 derivatives. Results presented here report for the first time by a new class of molecules that can become effective drugs against AD.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.     

Nanosecond dynamics of acetylcholinesterase near the active center gorge

To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible omega loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys(69)-Cys(96) omega loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.