Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom

This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin gamma is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in beta-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the beta-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the beta-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role, and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.     

Basic phospholipase from the venom of Naja nigricollis is cytotoxic

The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.     

Non-enzymatic inhibitors of coagulation and platelet aggregation from Naja nigricollis venom are cardiotoxins

Four non-enzymatic polypeptides from Naja nigricollis crawshawii venom were recently isolated and shown to inhibit plasma coagulation and platelet aggregation. We have now determined the amino acid compositions, amino terminal sequences and direct lytic activity of these anticoagulants. The results of these studies allow us to identify the anticoagulants as cardiotoxins. The anticoagulant activity of these cardiotoxins is far more potent than that of other cardiotoxins previously reported to have anticoagulant activity.     

Isolation and characterization of a cytotoxin P4 from the venom of Naja nigricollis nigricollis preferentially active on tumor cells.

Cytotoxin P4 was isolated from the venom of Naja nigricollis nigricollis in three steps and contained 55% of the crude cytotoxic activity. It had a molecular weight of 8 KD, was stable over a pH range of 1-11 and in boiling water for at least 15 min. It had no measurable enzymatic activities, but was destroyed by proteases. Concentrations of 0.8, 1, 1.2, 25. 20 and 45 ug/ml, were needed to destroy murine melanoma B16 and WEHI 3B leukemia, rat chondrosarcoma, mouse erythrocytes and spleen cells, and human erythrocytes, respectively, thereby showing preferential cytotoxicity to the examined tumor cells. It also prevented the development of the melanoma, leukemia and chondrosarcoma tumors in vivo when mixed with the cells prior to the injection into the animal.     

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.     

Effect of modification of tyrosine residues in cytotoxin-1 from Indian cobra venom. (Naja naja naja)

The role of tyrosine residues in the biological activity of cytotoxin-1 was evaluated using N-bromo succinimide. N-bromo succinimide effected the oxidation of tyrosine residues in cytotoxin-1 with an increase in absorption at 260 nm. N-chloro succinimide was ineffective in the oxidation of tyrosine residues in the toxin. Oxidation of a single tyrosine residue (at 3.50 equivalents of N-bromo succinimide/mole of the toxin) resulted in complete loss of lethal activity of the toxin. The lytic activity of the toxin (lysis of erythrocytes) remained uneffected even after three of the four tyrosine residues in the toxin were oxidised.     

Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.     

Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability

Chemical modification of lysine residues in two bacterial alpha-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 +/- 1 residues in BAA and 10 +/- 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.     

The oxidation of methionine and its effect of the properties of cardiotoxin VII1 from Naja melanoleuca venom.

Methionine residues 24 and 26 of cardiotoxin VII1 from Naja melanoleuca were oxidised to sulphoxides using N-chlorosuccinimide at pH 8.5. The number of equivalents of oxidant required for complete oxidation suggested that the methionine side-chains existed in a relatively "exposed" conformational state in cardiotoxin. The oxidised cardiotoxin was devoid of lethality. It was also non-haemolytic, both on its own and in the presence of phospholipase A2. However, it was still able to precipitate with anti-cardiotoxin antibody. CD studies indicated that the polypeptide backbone conformation was intact in the oxidised cardiotoxin but some perturbation of tyrosine residues was evident. The possibility of a direct or indirect involvement of the methionine residues in the biological activity of the cardiotoxin is discussed.     

Circular dichroism study of the unfolding-refolding of a cardiotoxin from Taiwan cobra (Naja naja atra) venom

Circular dichroism spectroscopy has been used to study the unfolding-refolding process of a cardiotoxin from Taiwan cobra (Naja naja atra) venom upon addition of fluoroalcohols or sodium dodecyl sulfate (SDS) to its aqueous solution. In these experiments, the disulfide bridges remained intact. The unfolding process has been found to be reversible both for fluoroalcohols and for SDS unfolding. The reversibility of the unfolding-refolding process of cardiotoxin in aqueous mixtures of fluoroalcohols was dependent on the volume per volume ratio of alcohol to water. SDS did not unfold the secondary structures of cardiotoxin whereas its tertiary structure was affected. If the SDS concentration in aqueous solution exceeded the critical micelle concentration value of SDS, a quasi-refolded state of cardiotoxin was observed. The mechanism of unfolding-refolding is discussed in terms of molecular interactions which might govern the protein conformation in solution.     

Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.     

The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra).

The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.     

Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study.

The solution conformation of toxin alpha from Naja nigricollis (61 amino acids and four disulfides), a snake toxin which specifically blocks the activity of the nicotinic acetylcholine receptor (AcChoR), has been determined using nuclear magnetic resonance spectroscopy and molecular modeling. The solution structures were calculated using 409 distance and 73 dihedral angle restraints. The average atomic rms deviation between the eight refined structures and the mean structure is approximately 0.5 A for the backbone atoms. The overall folding of toxin alpha consists of three major loops which are stabilized by three disulfide bridges and one short C terminal loop stabilized by a fourth disulfide bridge. All the disulfides are grouped in the same region of the molecule, forming a highly constrained structure from which the loops protrude. As predicted, this structure appears to be very similar to the 1.4-A resolution crystal structure of another snake neurotoxin, namely, erabutoxin b from Laticauda semifasciata. The atomic rms deviation for the backbone atoms between the solution and crystal structures is approximately 1.7 A. The minor differences which are observed between the two structures are partly related to the deletion of one residue from the chain of toxin alpha. It is notable that, although the two toxins differ from each other by 16 amino acid substitutions, their side chains have an essentially similar spatial organization. However, most of the side chains which constitute the presumed AcChoR binding site for the curaremimetic toxins are poorly resolved in toxin alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom

The cationic groups of arginine and lysine residues in-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) andp-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.     

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.     

Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex.

The strongly anticoagulant basic phospholipase A(2) (CM-IV) from Naja nigricollis venom has previously been shown to inhibit the prothrombinase complex of the coagulation cascade by a novel nonenzymatic mechanism (S. Stefansson, R. M. Kini, and H. J. Evans Biochemistry 29, 7742-7746, 1990). That work indicated that CM-IV is a noncompetitive inhibitor and thus it interacts with either factor Va or factor Xa, or both. We further examined the interaction of CM-IV and the protein components of the prothrombinase complex. Isothermal calorimetry studies indicate that CM-IV does not bind to prothrombin or factor Va, but only to factor Xa. CM-IV has no effect on the cleavage of prothrombin by factor Xa in the absence of factor Va. However, in the presence of factor Va, CM-IV inhibits thrombin formation by factor Xa. With a constant amount of CM-IV, raising the concentration of factor Va relieved the inhibition. The phospholipase A(2) enzyme inhibits by competing with factor Va for binding to factor Xa and thus prevents formation of the normal Xa-Va complex or replaces bound factor Va from the complex. Thus factor Xa is the target protein of this anticoagulant phospholipase A(2), which exerts its anticoagulant effect by protein-protein rather than protein-phospholipid interactions.