R. Manjunatha Kini

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

Significance Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey. Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

Anticoagulant proteins from snake venoms: structure, function and mechanism

Over the last several decades, research on snake venom toxins has provided not only new tools to decipher molecular details of various physiological processes, but also inspiration to design and develop a number of therapeutic agents. Blood circulation, particularly thrombosis and haemostasis, is one of the major targets of several snake venom proteins. Among them, anticoagulant proteins have contributed to our understanding of molecular mechanisms of blood coagulation and have provided potential new leads for the development of drugs to treat or to prevent unwanted clot formation. Some of these anticoagulants exhibit various enzymatic activities whereas others do not. They interfere in normal blood coagulation by different mechanisms. Although significant progress has been made in understanding the structure-function relationships and the mechanisms of some of these anticoagulants, there are still a number of questions to be answered as more new anticoagulants are being discovered. Such studies contribute to our fight against unwanted clot formation, which leads to death and debilitation in cardiac arrest and stroke in patients with cardiovascular and cerebrovascular diseases, arteriosclerosis and hypertension. This review describes the details of the structure, mechanism and structure-function relationships of anticoagulant proteins from snake venoms.

Accelerated Evolution and Molecular Surface of Venom Phospholipase A2 Enzymes

Abstract. Multiple phospholipase A2 (PLA2) isoenzymes found in a single snake venom induce a variety of pharmacological effects. These multiple forms are formed by gene duplication and accelerated evolution of exons. We examined the amino acid sequences of 127 snake venom PLA2 enzymes and their homologues to study in which location most natural substitutions occur. Our data show that hot spots of amino acid substitutions in this group of proteins occur mostly on the surface. A logistic model correlating the substitution rates of each amino acid residue with their surface accessibility indicates that the probability of natural substitutions occurring in the fully exposed residue is 2.6–3.5 times greater than that of substitutions occurring in buried residues. These surface substitutions play a significant role in the evolution of new PLA2 isoenzymes by altering the specificity of targeting to various tissues or cells, resulting in distinct pharmacological effects. Thus natural substitutions in PLA2 enzymes, in contrast to popular belief, are not random substitutions but appear to be directed toward modifying the molecular surface.

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle α1βγδ-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 Å by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snakes diet.

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Snake venoms are cocktails of enzymes and non‐enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l‐amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A2. Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure‐based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non‐enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor–enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.

Isolation of a Neurotoxin (α-colubritoxin) from a Nonvenomous Colubrid: Evidence for Early Origin of Venom in Snakes

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called α-colubritoxin, isolated from the Asian ratsnake Coelognathusradiatus (formerly known as Elapheradiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.

Molecular moulds with multiple missions: Functional sites in three‐finger toxins

1. Snake venoms are complex mixtures of pharmacologically active peptides and proteins.

Structure-function relationships of phospholipases I: Prediction of presynaptic neurotoxicity

The hydropathic indexes of 24 phospholipases have been calculated from their amino acid sequences. The presynaptic neurotoxic potential of venom phospholipases can be predicted by the use of hydropathy profiles. The presynaptically acting phospholipases have a distinct hydrophobic region around the residues 80-110 which is probably involved in the interaction with the presynaptic membranes. This region is present as a separate helix in the tertiary structure of phospholipases. Such a hydrophobic region is absent in non-neurotoxic phospholipases.

Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity

A novel heterodimeric three‐finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three‐finger protein fold, typical for “nonconventional” toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three‐finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon‐specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC50=10 nM), comparable to α‐bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three‐finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.—Pawlak, J., Mackessy, S. P., Sixberry, N. M., Stura, E. A, Le Du, M. H., Ménez, R., Foo, C. S., Ménez, A, Nirthanan, S., Kini, R. M. Irditoxin, a novel covalently linked heterodimeric three‐finger toxin with high taxon‐specific neurotoxicity. FASEB J. 23, 534–545 (2009)

From snake venom toxins to therapeutics--cardiovascular examples.

Snakes have fascinated the imaginations of people since the dawn of civilization. Their deadly venoms cause significant mortality and morbidity worldwide, and strike fear in most of us. Snake venoms contain a huge variety of molecules affecting vital physiological systems, and scientists are turning some of these life-threatening toxins into a source of life-saving therapeutics. Since the approval of captopril--the first drug based on snake venom protein--more than 30 years ago, snake venom toxins have become a valuable natural pharmacopeia of bioactive molecules that provide lead compounds for the development of new drugs. Many toxins are being explored and developed into drugs for the treatment of conditions such as hypertension, thrombosis and cancer. A number of new drugs are constantly emerging from this pipeline. In this review, we briefly highlight the molecular basis of developing therapeutic agents, such as Captopril, Tirofiban, and Eptifibatide, from snake venom proteins. We also discuss the successes and failures as an update to the advances in the field.