Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

Abstract

Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

Secreted Phospholipases A2 from Animal Venoms in Pain and Analgesia

“…CTX is a heterodimeric complex consisting of a non-covalent association between an acidic non-enzymatic protein (crotoxin A, CA or crotapotin) and a basic toxic phospholipase A 2 (crotoxin B or CB) [ 66 , 67 ]. CA is formed by three polypeptide chains (α, β and γ, where the α and β chains are α-helices with loops at the terminal positions, and the γ chain is a disordered loop) linked by disulfide bonds, whereas CB is a class II phospholipase A 2 [ 66 , 68 ]. CA is generated from a PLA 2 -like precursor (pro-CA) by the removal of three peptides, leaving unchanged the molecule core linked by disulfide bonds [ 69 ].…”

“…Apparently, the role of CA is to prevent CB adsorption to non-saturable binding sites, thereby restricting its binding to critical target sites at neuromuscular junctions [ 11 ]. In fact, it was recently demonstrated that titration of CA in CB tetramers causes the dissociation of CB oligomers, restoring the CTX heterodimer [ 68 ]. Furthermore, the inhibition of the catalytic activity of CB, as well as the enhancement in CB pharmacological activity caused by CA, suggest that other regions of CB, besides the active site, may be involved in its mechanism of action.…”

Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions

“…Crotoxin is a β neurotoxin, composed of two subunits: an active PLA 2 and the catalytic inactive crotapotin [ 22 , 23 ]. Since most venoms and toxins present isoforms, a consequence of an evolutionary strategy, we have chosen to evaluate whether there would be a preferred substitution site in a given crotapotin subunit that would give rise to the previously observed isoforms [ 21 ].…”

“…Such mutation is located in an α helix and the introduction of a charged residue may alter such structure. Moreover, this particular region is mostly involved in the PLA 2 interaction [ 23 ].…”

Heterologous Expression and Purification of Recombinant Crotoxin B, the Phospholipase A2 Subunit of Crotoxin

“…a Crotoxin is a heterodimeric protein that consists of a non-toxic, nonenzymatic, acidic subunit (crotapotin, crotoxin A, CA) non-covalently associated with a weakly toxic, basic subunit (crotoxin B, CB) that expresses phospholipase A 2 activity. The CA subunit acts as a chaperone protein, potentiating the toxicity of the CB subunit [1,3]. Four different isoforms have been identified for each subunit, consequently the venom of Crotalus durissus terrificus contains up to 16 different crotoxin complexes, each presenting different activity levels [4,5].…”