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Identification of a ?-helical molten globule intermediate and structural characterization of ?-cardiotoxin, an all ?-sheet protein isolated from the venom of Ophiophagus hannah (king cobra).

ABSTRACT: ?-Cardiotoxin is a novel member of the snake venom three-finger toxin (3FTX) family. This is the first exogenous protein to antagonize ?-adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all ?-sheet peptides with 60-80 amino acid residues. Here, we describe the three-dimensional crystal structure of ?-cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of ?-cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of ?-helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of ?-cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.

SUBMITTER: Roy A

PROVIDER: S-EPMC6459992 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Identification of a α-helical molten globule intermediate and structural characterization of β-cardiotoxin, an all β-sheet protein isolated from the venom of Ophiophagus hannah (king cobra).

Roy Amrita A Qingxiang Sun S Alex Chapeaurouge C Rajagopalan Nandhakishore N Jobichen Chacko C Sivaraman J J Kini R Manjunatha RM

Protein science : a publication of the Protein Society 20190404 5

β-Cardiotoxin is a novel member of the snake venom three-finger toxin (3FTX) family. This is the first exogenous protein to antagonize β-adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β-sheet peptides with 60-80 amino acid residues. Here, we describe the three-dimensional crystal structure of β-cardiotoxin together with the identification of a molten glo ...[more]

PMID: 30891862

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Project description:BackgroundThe king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS.ResultsTranscriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A2 (PLA2s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). Seventeen toxin families exhibited low mRNA expression, including hyaluronidase, DPP-IV and 5'-nucleotidase that were not previously reported in the venom-gland transcriptome of a Balinese O. hannah. On the other hand, the MOh proteome includes 3FTxs, the most abundantly expressed proteins in the venom (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 non-conventional 3FTx. Neurotoxins comprise the major 3FTxs in the MOh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. The presence of toxic enzymes such as LAAOs, SVMPs and PLA2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) are in agreement with the poor cross-neutralization activity of N. kaouthia antivenom used against MOh venom. Besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5'-nucleotidase, and DPP-IV in the venom proteome suggests its probable hypotensive action in subduing prey.ConclusionThis study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). The results correlate with the pathophysiological actions of MOh venom, and dispute the use of Naja cobra antivenoms to treat MOh envenomation. The findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom.

Project description:BackgroundBeta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein.Methodsβ-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration.ResultsPurified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis.ConclusionWe present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX.

Dataset Information

Identification of a ?-helical molten globule intermediate and structural characterization of ?-cardiotoxin, an all ?-sheet protein isolated from the venom of Ophiophagus hannah (king cobra).

Publications

Identification of a α-helical molten globule intermediate and structural characterization of β-cardiotoxin, an all β-sheet protein isolated from the venom of Ophiophagus hannah (king cobra).

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