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Evolutionary Divergence of Scorpion Toxins: Nature and Mechanism of Action: A Computational Analysis

R. K. Upadhyay and R. N. K. Bamezai

School of Life Sciences & Bioinformatics Center, JawaharLal Nehru University, New Delhi 110 067, India

Conserved and consensus sequences of several members of scorpion toxin families were analyzed. Multiple sequence alignment defined the highly conserved residues that play important structural role in toxin sub group/families. For  conformation  analysis, toxins were classified according to their maximum homology examined from their amino acid sequences and according to their nature of active site region.

Scorpion toxins have characteristic feature in signature pattern {(GA)}-K-C(LIVM)-x(2)-K-C-Cx-C)}, where lysines and cysteines are well conserved in the toxin polypeptide. The toxins show two major groups, one with the conserved region GKCMNKCDC and a second with CTXPK.   There is variability in the position of lysine and arginine in the different toxins. Most of the scorpion toxins contain six conserved cysteines involved in disulfide bonds. These regions act as blockers of calcium and sodium channels.

Functionally, in scorpion toxins, there are some minor changes leading to lower activity as sodium channel blockers, when replacement of lysine by arginine takes place. Same phenomenon is apparent with lysine replacement by phenyalanine and serine in neurotoxins.   In the scorpion family among signature sequences most of the residues are conserved in neurotoxins having nearly same conserved residues in the active site region. Neurotoxins are either sodium channel inhibitors or voltage gated sodium channel blockers. Sodium channel binding domain in neurotoxins have residues GKCMNRKC with first and second lysine and cysteine well conserved. Active site region in charybdotoxin, tityustoxin and agitoxin is well conserved between 26-33 amino acid residues while kaliotoxin (SCK2_Andma) is an exception having active site region between 47-54 amino acids. Interspecific homologies also indicate that scorpion signatures are also partially shared by small domains of defensin, phosphorylase and integrins of plants.

Homology identification of close associates in evolution was made by analyzing amino acids from primary sequences. Agitoxin studied exhibited an excellent conformational homology with leiurotoxin but consensus sequence of kaliotoxin (KTX) had some difference in homology at N terminal side. CTPK of kaliotoxin is well conserved in agitoxin and leiurotoxin. Noxiustoxin has high homology with mergotoxin while tityustoxin and neurotoxin are a different group with same conserved sites between 50-60 amino acids. GYC is  the characteristic  triplet between both the proteins. Insectoxin is  a neurotoxin in N terminal side. Starting from the leiurotoxin many  substitutes of basic amino acids and replacements of  amino acids  with  similar basic active amino acids might be  a  strong point  for  channel  binding. From the Leuirus  species, so many toxins were formed in evolution which are present in the same species that are chlorotoxin, insectoxin, neurotoxin, agitoxin and leiurotoxin from the single species. How this species has  so much diversification in the nature of toxin is a very  important question for evolutionists.

In detailed homology analysis, the presence and number of lysines in active site region as well as in alpha helical region determine the nature and level of toxicity. In particular, Lys 27 of charybdotoxin was a critical residue which could bind to the ion conduction  pathway of the channel which was also seems  to  have important role in kaliotoxin, margotoxin, insectoxin and  leiuro-toxin.  Basic amino acids replaced by some  non-charged polar groups may be responsible for non-target of channel binding or  they may be transformed into a less toxic polypeptide. High-grade killer toxins contain highly charged basic amino acids in the alpha helical region. In regular evolution, G, K, C, M, N, H, C and R  were the most conserved residues which have shown high  activity  for channel binding which proved the presence of highly basic  amino acids are strong evolutionary species.

From evolutionary analysis, Leuirus is a most primitive ancestral species from which various toxins were originated. Among toxins, neurotoxins are the most diversified  toxins  which are available from temperate to colder regions, but its basic nature is  also the same, which really indicates that the conserved active site  region from the signature sequences of neurotoxins is  most constant  phenomena throughout the long evolution which was  not changed in so much time period. This point led to the conclusion that from Leiurus neurotoxins evolved first and through shifting mechanism other different toxins came into the existence.

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