Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin.

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.

A stable miniature protein with oxaloacetate decarboxylase activity.

An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.

Defining determinant molecular properties for the blockade of the apamin-sensitive SKCa channel in guinea-pig hepatocytes: the influence of polarizability and molecular geometry.

QSAR studies of a series of blockers of the SK(Ca) channel in guinea-pig hepatocytes suggests that the polarizability of the blocker is an important factor controlling the binding to the channel. It is suggested that, upon binding, an ion-pair is formed, a process that is promoted by the reorganization of the water molecules. The polarizability is not adequate to describe the potency of the most potent blockers with a good stereochemical fit to the channel, presumably due to more specific interactions taking place.

Synthesis and conformational analysis of apamin analogues with natural and non-natural cystine/selenocystine connectivities.

By replacing two cysteine residues in apamin with selenocysteine, the three possible isomers related to the side-chain connectivities of a bis-cystinyl-peptide were synthesized in regioselective manner exploiting the low redox potential of the diselenide bond. Nuclear magnetic resonance conformational analysis of monoselenocystine analogue apamin with the natural diselenide/disulfide network confirmed the highly isomorphous character of the sulfur replacement with selenium despite its slightly larger atomic radius and increased bond lengths. The comparative conformational analysis of the apamin analogues containing the non-natural side-chain links with wild type apamin clearly revealed retention of the main structural fold and thus the high propensity of these small molecules to adopt the secondary structure elements present in natural apamin. These findings offered interesting hints for a better understanding of the oxidative refolding pathway of the bis-cystinyl peptide that leads exclusively to the correct natural isomer.

Photolabile derivatives of 125I-apamin: defining the structural criteria required for labeling high and low molecular mass polypeptides associated with small conductance Ca(2+)-activated K+ channels.

The structure of apamin-sensitive Ca(2+)-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha-formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino-Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30-kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin-sensitive Ca2+-activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.

Recombinant and chemical derivatives of apamin. Implication of post-transcriptional C-terminal amidation of apamin in biological activity.

The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col-(1-171)-apa] and HPLC purification. It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue. Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non-amidated C-terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity.

One-disulfide intermediates of apamin exhibit native-like structure.

The conformations of three peptide models of the one-disulfide and fully reduced forms of apamin were characterized by using nuclear magnetic resonance (NMR) spectroscopy. Apa-2 contains the native disulfide bond between Cys3 and Cys15 in apamin with the other two cysteines replaced by alanines. Apa-1 contains the native disulfide bond between Cys1 and Cys11. Apa-S has all cysteines replaced with serines, mimicking fully reduced apamin. Comparing NOESY cross peaks and coupling constants for amide protons in the peptide analogs with those in native apamin indicates that a significant portion of Apa-2 possesses native-like structural elements of apamin in addition to some random coil conformations. Apa-1 contains a short helical structure from Ala9 to Arg13, corresponding to the N-terminal portion of the alpha-helix observed in the native structure, along with some local and probably flexible secondary structures corresponding to the reverse turn region in native apamin. A larger portion of Apa-1 exists in the form of random coil conformations compared to Apa-2. Apa-S displays mainly random coil conformations with some localized helical structures from Glu7 to Arg14 which are similar to the "nascent helices" proposed by Wright et al. [Wright, P. E., Dyson, H. J., & Lerner, R. A. (1988) Biochemistry 27, 7167-7175]. Formation of the first disulfide bond in apamin seems to be important in the folding process by stabilizing native-like structure, presumably by reducing the conformational freedom and initiating formation of structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Solution conformation of leiurotoxin I (scyllatoxin) by 1H nuclear magnetic resonance. Resonance assignment and secondary structure.

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulfide bridges locate the N-terminal part (that is alpha-helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel beta sheet (from Leu18 to Val29). The latter features a tight turn at Gly23-Asp24.

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide.

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes.

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.

Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

An azidonitrophenylaminoacetyl mono[125I]iodoapamin derivative was prepared which showed specific binding to rat neuronal membranes. UV photolysis lead to the irreversible occupation of binding sites. Photo-labeling of intact primary cultured rat neurones followed by membrane solubilization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography revealed the covalent incorporation of radioactivity into 3 main components with Mr = 86,000, 30,000, and 23,000. Labeling was completely prevented by a competing excess of native apamin. Similar studies on purified synaptic membranes from the rat brain showed another labeling pattern with major bands corresponding to Mr = 86,000 and 59,000. Although the reasons for the partial discrepancy between cultured embryonic neurons and an adult brain membrane fraction are not yet clear, we conclude that these proteins are intimately associated with the apamin binding site and are probably components of a type of Ca2+-activated K+ channel.

Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes.

The bee venom peptide, apamin, has been radiolabelled with 125I, the monoiodinated derivative purified, and its binding to intact guinea-pig liver cells studied. At 37 degrees C 125I-monoiodoapamin associated with, and dissociated from, guinea-pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 X 10(8) M-1 s-1 and 0.035 s-1 respectively. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pM. The maximal binding capacity was 1.1 fmol mg-1 dry wt. of tissue. Unlabelled apamin displaced bound 125I-monoiodoapamin with a KI of 380 pM, which is consistent with the concentration of apamin required to inhibit Ca2+-activated K+ permeability (PK(Ca) ) in these cells. Inhibitable binding of 125I-monoiodoapamin to rat hepatocytes was much less than to guinea-pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the reported lack of apamin-sensitive Ca2+-activated K+ channels in these cell types. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+-mediated K+ efflux from, guinea-pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA and quinine affected hepatocytes only at high concentration (KI = 5.8 and 0.51 mM respectively). 9-aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70-180 microM). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7.5, 6.8 and 4.5 microM respectively). Gallamine was slightly less effective (KI = 14 microM) and decamethonium and hexamethonium much less so (KI = 620 and 760 microM respectively). 3,4-diaminopyridine, alpha-bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea-pig hepatocytes. The saturable binding of 125I-monoiodoapamin to guinea-pig hepatocytes corresponds to about 1700 sites per cell. Assuming, tentatively, that binding sites correspond to channels the rate of K+ loss observed following agonist action can readily be explained if these channels have unitary conductances in the range reported for PK(Ca) in other tissues.

High affinity binding of [125I]monoiodoapamin to isolated guinea-pig hepatocytes.

The bee venom neurotoxin apamin has been labelled with 125I and its binding to isolated guinea-pig hepatocytes measured under physiological conditions. A single saturable component of [125I]monoiodoapamin binding with a Kd of 350 pM and Bmax of 0.99 fmol/mg dry wt was identified. Native apamin displaced labelled apamin with a Kd of 376 pM which is broadly in keeping with the concentrations found to inhibit K loss from guinea-pig hepatocytes. These observations, together with the binding found in other tissues, suggest that specific binding of labelled apamin is particularly associated with apamin-sensitive, Ca-activated K-channels.

Preparation of a pure monoiodo derivative of the bee venom neurotoxin apamin and its binding properties to rat brain synaptosomes.

The preparation and purification of an active monoiodo derivative of apamin is described. Radiolabeled monoiodoapamin (2000 Ci/mmol) binds specifically to rat brain synaptosomes at 0 degrees C and pH 7.5 with a second order rate constant of association (ka = 2.6 x 10(7) M-1 s-1) and a first order rate constant of dissociation (kd = 3.8 x 10(-4) s-1). The maximal binding capacity is 12.5 fmol/mg of protein and the dissociation constant is 15-25 pM for the monoiodo derivative and 10 pM for the native toxin. The apamin receptor is destroyed by proteases suggesting that it is of a proteic nature. Neurotensin and its COOH-terminal partial sequences are the only molecules unrelated to apamin that are able to displace monoiodoapamin from its receptor at low concentrations. Half-displacement occurs at 170 nM neurotensin. This property is due to the presence in the COOH-terminal sequence of neurotensin of two contiguous arginine residues, a structure analogous to that of the apamin active site. The binding of monoiodoapamin to its receptor is sensitive to cations. Increasing K+ or Rb+ concentrations from 10 microM to 5 mM selectively enhances the binding by a factor of 1.8. Increasing the concentration of any cation from 1 to 100 mM completely inhibits iodoapamin binding. Both effects are due to a cation-induced modulation of the affinity of monoidoapamin for its receptor without any change of the maximal toxin binding capacity of synaptosomes. Guanidinium and molecules containing a guanidinium group are better inhibitors of iodoapamin binding than other inorganic cations or positively charged organic molecules.

Solid phase synthesis of 13-lysine-apamin, 14-lysine-apamin and the corresponding guanidinated derivatives.

In order to study the importance of arginine residues 13 and 14 in apamin, the bee venom neurotoxin, four analogues, [Lys13]-apamin, [Lys14]-apamin, [Har4, Har13]-apamin and [Har4, har14]-apamin were synthesized and tested with respect to their neurotoxicity. The two lysine-apamins were prepared by the solid phase method on benzhydrylamine resins. Before oxidation to disulphides, the (S-Acm)4-peptides were isolated and characterized. Portions of the purified lysin peptides were converted to homoarginine analogues by guanidination. The four apamin analogues were lethal, but the lethal doses differed significantly. The results demonstrate that the arginine residue at position 14 is more important for the high toxicity than is the one at position 13. The circular dichroism (CD) spectrum of [Lys13]-apamin was identical with that of apamin itself, whereas the spectrum of [Lys14]-apamin showed certain deviations.

Use of synthetic analogs for a study on the structure-activity relationship of apamin.

[Lys13,Lys14]Apamin, [Lys13]apamin and [Lys14]apamin, three structural analogs of the bee venom neurotoxin, have been obtained by solid-phase peptide synthesis while an attempt to obtain [Cit13]apamin failed, probably at the step of reoxidation of cysteines. After the chemical purity of these three derivatives had been assessed, further chemical modifications led to three new peptides: [Ac-Cys1,Lys(Ac)4,Lys(Ac)13]apamin, [Ac-Cys1,Lys(Ac)4,Lys(Ac)14]apamin and [Har4,Har13,Har14]apamin. These six analogs have been tested for their neurotoxicity, i.e. determination of LD50 for mouse by subcutaneous injection. A lethal potency is observed only when the region 13-14 of the sequence contains a double positive charge. One arginyl residue is necessary for a high biological activity, while its location in position 13 or 14 is of minor importance. When homoarginine (Har) replaces arginyl residues the neurotoxicity is lowered.