Buried in sand in the warm waters of the Bay of Bengal, a predator waits. As a fish darts past, a tiny harpoon tipped with deadly venom flashes by and buries itself in the fish. The, predator, a marine cone snail of the genus Conus glides towards the paralysed, weakly twitching fish and engulfs it. It’s all over in a matter of seconds. The potent neurotoxic venoms of Conus snails, called conotoxins, are hot topics for research since they have immense potential as pain relief agents or analgesics (think very strong, but non-addictive morphine). Researchers from the Molecular Biophysics Unit at the Indian Institute of Science, Bangalore, have added their mite to this fascinating field. They have figured out the 3-dimensional structure of a conotoxin named Mo3964 from the venom of the cone snail Conus monile.
“It’s an amazing molecule with a unique structure”, says Aswani Kancherla, who is the lead author in a paper describing the work on Mo3964, which was recently published in the prestigious journal ACS Chemical Biology. Mo3964 is a small protein, called a peptide, made of 37 amino acids (which are the basic building blocks of proteins); it is rich in an amino acid called cysteine.
What’s so special about this? Unlike most other amino acids, cysteine contains sulphur as a thiol group (-SH). Very often, in proteins and peptides, the thiol groups from two cysteines react to form a disulphide bond (-S-S-). Frequently, these bonds are crucial for proteins to maintain their structure, which in turn, is prerequisite for them to function correctly.
“The pattern of disulphide bonds in Mo3964 makes this molecule incredibly stable”, states Aswani. The key folding pattern that imparts an astonishing degree of stability to Mo3964 involves two of the disulphide bonds that form a structure which looks like two crossed hands. “Dr. Srinu Meesala (a co-author) and I had a difficult time obtaining purified peptide from bacterial cultures using the cloned gene, but I didn’t expect it [Mo3964] to be so difficult to break down”, Aswani marvels. “Enzymes didn’t affect it, and even heating upto 80°Cleft it pretty much unchanged”, he adds with a laugh. But when the disulphide bonds are broken using chemicals called “reducing agents”, Mo3964 falls apart and unfolds quickly even at room temperature.
Why is the 3-D shape of a molecule so important? Because, armed with this knowledge, scientists can actually predict the effect of a drug or the function of a protein and can design newer and better drugs or proteins for medical use. The 3-D structure of Mo3964 was solved using NMR (Nuclear Magnetic Resonance), a technique that uses the magnetic properties of certain atoms to provide information on the shape, chemistry and dynamics of molecules. X-ray crystallography, which is mostly used to study 3-D molecular structures of proteins, needs proteins in a crystallised form and only provides a “freeze-frame” version of a molecule’s shape. “The main advantage of using NMR, is that you don’t need to get protein crystals and you can study them [proteins] in solution, which is how they normally exist”, says Aswani.
What makes Mo3964 even more exceptional, is that the folding pattern of this peptide has never before been described in conotoxins. Aswani is excited about this aspect of the molecule’s distinctiveness. “It’s a new bioactive peptide fold! New structural patterns like these are always important. We can use them to design novel peptides or drugs with different functions”, he says. Conotoxins are emerging as important sources of novel drugs because of two main reasons – one, they are more selective in acting on specific targets than generic ‘small molecule drugs’ (like aspirin, whose mechanism of pain suppression is still unknown); and two, they are much more stable than ‘biological protein drugs’ like antibodies. Preliminary studies by Aswani’s co-authors, Pooja Jorwaland Ramasamy Palanisamy on the biological activity of Mo3964 show that it may affect nerve cells by impacting the function of potassium and sodium ion channels.
Every snail species generally has a unique cocktail of neurotoxins, to help it to hunt worms, fish or even other snails, which are its prey. Mo3964 is just one toxin from a battery of about 100 – 200 toxins in the venom of the snail Conus monile. Its name is a label indicating that it comes from the species C. monile (the Mo part), and the number (3964) indicates its molecular weight. The incredible variety of conotoxins makes this group of biomolecules a rich source of potential neuro-active drugs. Currently, few conotoxins (Ziconotide and Leconotide, to name two) are being investigated as potential therapeutic drugs for managing extreme and chronic pain conditions. Despite their high efficacy in treating pain, difficulties in administering these conotoxins as well as severe side effects have hampered their widespread use. Future research on the structure and function of newer conotoxins, will hopefully provide us with more promising candidates for addiction-free pain medication.
About the authors
Siddhartha Sarma and Sujit Sikdar are professors at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore. Aswani K. Kancherla , Srinu Meesala , Pooja Jorwal and Ramasamy Palanisamyare research scholars in their labs.
About the paper
The paper appeared in ACS Chemical Biology on 11 May. http://pubs.acs.org/doi/abs/10.1021/acschembio.5b00226