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.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex βγ-CAT evolved to drive cell vesicular delivery and transport

“…Liposomes were composed of phosphatidyl choline (100% PC). The method was mainly referred as references ( 70 , 71 ) with some modifications. PC (Picasso) was dissolved in a methanol/chloroform mixture at a 1:9 ratio with a final concentration of 35 mg/ml and protected from light in distillation flasks.…”

Fifty Years of Animal Toxin Research at the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

“…The first, and so far unique, among three-fingered toxins, glycosylated cytotoxin was isolated from cobra N. kaouthia venom, where the glycosylation of Asn29 in loop II resulted in a decrease in its biological activity [ 9 ]. It was found that the cytotoxicity depended on the structure of loops I and II, the toxins with two adjacent proline residues in loop I being the least active [ 78 , 79 ]. Using the confocal spectral imaging technique and fluorescently labeled cytotoxins, it was shown that cytotoxins penetrated readily into cells and accumulated markedly in the lysosomes, and this accumulation correlated well with the cytotoxic effects [ 79 ].…”

Non-bilayer Lipid Phases Modulate the Structure and Functions of Mitochondrial Membranes: Pharmacological Relevance

“…Copyright© Edward S Gasanoff Cytotoxins CTI and CTII, membrane-active cationic proteins of 7 kD from the Central Asian cobra venom, have been widely used in studies to probe the structural organization of model lipid membranes [21,45,[57][58][59][60][61][62][63][64][65][66][67]. Both cytotoxins phenocopy membranotropic properties of C8 subunit of the F0 sector in bovine ATP synthase [9,61].…”

“…It has been shown that CTII decreases the angular anisotropy of the spin-label EPR spectral signals in lipid films enriched with acidic phospholipids, while CTI demonstrates the same ability only in lipid films enriched with CL, but not with other acidic phospholipids [59]. Both CTI and CTII trigger aggregation of liposomes and dehydration of the membrane surface [45,[57][58][59][60][61]. CTII induces intermembrane exchange of lipids and membrane fusion in membranes enriched with any kind of acidic lipids, while CTI is able to do the same only in membranes enriched with CL [45,[57][58][59][60][61][65][66][67][68].…”

“…Both CTI and CTII trigger aggregation of liposomes and dehydration of the membrane surface [45,[57][58][59][60][61]. CTII induces intermembrane exchange of lipids and membrane fusion in membranes enriched with any kind of acidic lipids, while CTI is able to do the same only in membranes enriched with CL [45,[57][58][59][60][61][65][66][67][68]. 31P-and 1H-NMR spectroscopy studies of the large unilamellar liposomes made of either PC+10mol% CL or PC+10mol% PS and treated with either CTI or CTII have shown that CTII induces the formation of non-bilayer structures and increases membrane permeability of both PC+10mol% CL and PC+10mol% PS liposomes, while CTI induces formation of non-bilayer structures and increases membrane permeability only in PC+10mol% CL liposomes (Figure 2) [59,61].…”