Structural and functional characterization of suramin-bound MjTX-I from Bothrops moojeni suggests a particular myotoxic mechanism

Guilherme H M Salvador,Marcos R M Fontes,Mário De Oliveira Neto,César A Gandin,Antoniel A S Gomes,Juliana I Dos Santos,Walter L G Cavalcante,Thiago R Dreyer,Márcia Gallacci

doi:10.1038/s41598-018-28584-7

Guilherme H M Salvador, Marcos R M Fontes + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-28584-7

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Abstract

Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.

Highlights

Ophidian accidents represent an important public health problem in rural areas of Asia, Africa and Latin America, where the number of deaths caused by snakebites are higher than other neglected tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease
We studied the binding behaviour between MjTX-I, a myotoxic phospholipases A2 (PLA2)-like isolated from Bothrops moojeni venom, and the suramin molecule using a broad combination of techniques, including myographic assays, molecular dynamic simulations, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC) and X-ray crystallography
After pre-incubation with suramin (125 μM) for 15 minutes, MjTX-I did not depress the twitch amplitudes; instead, it induced a discrete but significant facilitation of twitches starting at 75 minutes of toxin contact

Summary

Introduction

Ophidian accidents represent an important public health problem in rural areas of Asia, Africa and Latin America, where the number of deaths caused by snakebites are higher than other neglected tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease These facts lead the World Health Organization[1,2,3] to classify snakebites as a neglected disease, increasing the interest of the scientific community to study the compounds of these venoms and their eventual neutralization by specific inhibitors. Some of these regions comprise the myotoxic site (Lys[20], Lys[115] and Arg118) — subsequently called the membrane docking site (MDoS) — and the membrane disruption site (MDiS) composed of the conserved residues Leu[121] and Phe12533 Despite these hypotheses being applied to the majority of bothropic PLA2s-like toxins, MjTX-I is an exception[34] because its sequence does not present all the MDoS residues, and its native crystal structure exhibits a different oligomeric configuration. These factors were noted as possible factors for MjTX-I reduced myotoxicity when compared to other PLA2-like toxins[34]

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