Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop

Abstract

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

Keywords: antibacterial activity; biological membrane; calcein leakage; cobra cytotoxin; cytotoxic activity; phospholipid liposomes; spatial structure.

Figure 1

Conformational equilibrium of CTX in aqueous solution. (a) Major (left, pdb code 1CB9) and minor (right, 1CCQ) forms of cytotoxin 2 from N. oxiana in an aqueous solution. The major form is characterized by a trans-configuration of the Val7-Pro8 peptide bond while in the minor form this bond is in a cis-configuration (shown below). The N and C-termini are marked with N and C, respectively. The loops are numbered with Roman numerals. (b) For a toxin γ from N. nigricollis, only one form (pdb code 1TGX) with a cis-configuration between Pro8-Pro9 residues is present (this bond is shown below) (c) The equilibrium depends on the amino acid sequence within the tip of loop-I. The fragment of amino acid sequence of cytotoxin 2 from N. oxiana (upper sequence) is compared with that of toxin γ (lower sequence) from N. nigricollis. The substitution of the 9th residue to Pro (residues 8–9 are enclosed in the box) stabilizes the cis form. On all the panels only backbone atoms are shown. The orientation of all the molecules is identical. The side-chains in the inserts below the conformers (a, b) are shown in thin line, while backbone is in bold line. All inserts are magnified.

Figure 2

Amino acid sequences of the isolated toxins and elements of their secondary structure. The positions of disulfide bonds (S-S), beta-strands (rectangles with β inside) and extremities of the loops (double-headed arrows between rectangles) are indicated under the amino acid sequences. The numbers above the arrows correspond to the numbers of the first and the last amino acid residues. The numbering of the amino acid residues for all sequences is shown above. The residue Ser-28 and Pro-30, according to the presence of which CTX are classified as S- and P-type, respectively, are enclosed into boxes. The N. haje toxins with two prolines (indicated below the amino acid sequences) in loop-I are separated by the horizontal line from N. naja toxins with a single Pro residue in this loop. Similarity between the amino acid residues of the toxins is shown by color.

Figure 3

Spatial structures of the N. naja and N. haje CTX. In structures of N. naja toxins: 17–3 (a), 16–1 (b), 14–1 (c), and 15–1 (d), as well as N. haje toxins Nh1 (e)and Nh2 (f), only backbone and side-chains (heavy atoms) of the charged (positively charged Arg, Lys–blue, negatively charged Asp, Glu –red) amino acid residues are shown. In panel (a), the loops are numbered with Roman numerals. The orientation of CTX molecules in all the panels is identical, so the loop numbering is the same as in panel (a). In addition, C and N-termini of the molecules are marked. The backbones of the molecules are colored grey in all the panels. Note that the N. naja toxins and N. haje toxins are different primarily in the organization of loop-I (see Figure 1 for details).

Figure 4

Response of B. subtilis to increasing concentrations of CTX from N. naja venom. For each concentration, a set of 5 bars of distinct color is shown. The experiments for CTX Nh1, Nh2 from N. haje venom, featuring a Pro-Pro bond in loop-I, did not reveal any activity in the studied concentration range.

Figure 5

Dependence of A549 cell survival on the concentrations of CTX from N. naja venom after 3 h of incubation. The black curve corresponds to the control experiment, where no CTX was added.

Figure 6

Time-dependence of calcein leakage from DOPC/DOPG (1:1) unilamellar liposomes induced by CTX. The concentration of the total lipid was 0.1 mM. The concentration of added CTX was 1 µM. The vertical bars represent an experimental error, estimated by averaging after three measurements. The black curve corresponds to the control experiment, where no CTX was added to the liposomes.

Figure 7

Conformational equilibrium of CTX with a single Pro residue in loop-I in aqueous solution and their binding to a lipid membrane. The numbers in circles below the models correspond to the structural states of cytotoxin 1 from N. oxiana (CT1No) in an aqueous solution and lipid membrane. The structures were determined by NMR spectroscopy in an aqueous solution and detergent micelles. Only backbone is shown for all the structural models. The pdb code of state 1 determined for the recombinant CT1No is 5LUE (Met0-residue was removed). The bond Val7-Pro8 in state 1 is in cis-configuration, and trans-configuration in all other models (2–5) (see also inserts in Figure 1a, for the details of the conformation of this bond). The loops are marked with Roman numerals for state 1. The numbering of the loops is from left to right for all other models. States 2 and 5 are models 5NPN and 5NQ4, respectively. The membrane-interacting loops of the models, corresponding to states 3–5, are marked in yellow. The membrane surface is schematically shown with a horizontal line. The transition from state 3 to 4 is accompanied with conformational changes within loop-II of the molecule.