Structural Difference between Group I and Group II Cobra Cardiotoxins: X-ray, NMR, and CD Analysis of the Effect of cis-Proline Conformation on Three-Fingered Toxins

Abstract

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues. By binding to the membrane environment, group I CTX undergoes a conformational change to adopt a more extended hydrophobic domain with beta-sheet twisting closer to the one adopted by group II CTX. This result resolves a discrepancy in the CTX structural difference reported previously between solution as well as crystal state and shows that, in addition to the hydrophobicity, the exact loop I conformation also plays an important role in CTX-membrane interaction. Potential protein targets of group I CTXs after cell internalization are also discussed on the basis of the determined loop I conformation.

Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption

“…It is interesting that CTX A6, a CTX homolog identified only cobras caught in the eastern part of Taiwan (54), exhibited weak ␣v␤3 binding activity. CTX A6 was not cytotoxic in cardiomyocytes, CHO-K1 cells, and H9C2 myoblasts.…”

“…It is interesting to point out that the loop I conformation of CTX A6 adopts a type VI turn with a cis-peptide bond between the two prolines of the conserved Leu-Ile-Pro-Pro-Phe sequence for group I CTXs (54). In contrast, CTX A5 was previously classified as a group II CTX based on the absence of a proline residue at position 9.…”

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation

“…Many studies have been performed to shed light on the structure and function relationships of cobra venom toxins. The three-dimensional structures of most toxic components, including NTXs [9], PLA2s [10], CTXs (with different homologues of A1, A2, A3, A4, A5 and A6) [11], snake venom metalloproteinase (SVMP; K-like and atragin) [12], cysteine-rich secretory protein (CRISP) [13], and venom nerve growth factor (vNGF) [14] from Naja atra, are available. In addition, the genome from the king cobra (Ophiophagus hannah) [15], transcriptome from N. atra [16], and proteomes from African (Naja mossambica, Naja nigricollis, Naja katiensis, Naja nubiae, Naja pallida, and Naja haje) [17] and southeast Asian cobras (Naja kaouthia and Naja sumatrana) [7,18] have also been reported recently.…”

“…This provides us with a unique chance to collect venom samples from individual snakes. In fact, we previously reported a novel CTX A6 detected in cobra venom collected from the eastern region and determined its three-dimensional structures [11]. Although the potency of the newly identified toxin is not clear, this discovery raised an important issue as to whether commercially available antivenom could effectively neutralize its potential toxicity [7,34,35,38].…”

Endocytotic Routes of Cobra Cardiotoxins Depend on Spatial Distribution of Positively Charged and Hydrophobic Domains to Target Distinct Types of Sulfated Glycoconjugates on Cell Surface

“…The specific targets of demonstrated cell toxicity have been shown to be cell surface carbohydrates of glycosaminoglycans (GAG), membrane lipids of sulfatides, and/or membrane proteins of integrin, depending on the biological assay used for the investigations (1-3). The exact biological reasons for the diverse targets of the various CTX homologues are not clear, although it has been demonstrated that the toxin levels of cobra venom proteins exhibit significant geographic variation for Taiwanese cobras caught in the wild (4). For instance, CTX A2 and A4 toxins are present in cobra venoms from the western coast but are absent in the snakes from the eastern coast.…”