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

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.

Structural basis for phospholipase A2-like toxin inhibition by the synthetic compound Varespladib (LY315920)

“…The activation of PLA 2 -like toxins involves important structural changes in their oligomeric structures with particular features after the binding of fatty acids into the solvent-exposed hydrophobic channel: ( i ) alignment of the membrane docking site (MDoS), where the distance between positive clusters that comprise the MDoS (Lys16, Lys20, Lys115 and Arg118) from both monomers must be ≤16 Å; ( ii ) Euler Roll angle >160° and; ( iii ) changing the distance of the MDiS hydrophobic residues Leu121 (122 for MjTX-II) and Phe125 ≤ 5 Å 21,60 . These structural features were observed in recent studies by analyses of several crystallographic structures of PLA 2 -like toxins in native form and bound with inhibitory molecules 6,25,37,41,44–46,49,50,61 .…”

The allosteric activation mechanism of a phospholipase A2-like toxin from Bothrops jararacussu venom: a dynamic description

“…BthTX-I is a model protein in the group of PLA 2 -like proteins, which are mainly called myotoxins because of their myotoxic effect and other toxic effects by their membrane destabilization activity 14,23,38 . It has been shown that these proteins may assume a dimeric conformation when in solution 9,18 ; in other cases, their degree of oligomerization is concentration-dependent 39 , or related to the binding of particular ligands 20,40 . Furthermore, it is important to emphasize that their activity on the membrane is dependent on dimeric conformation 15,41 .…”

“…Indeed, studies of the molecular mechanisms for the inhibition of PLA 2 -like toxins with different molecules have confirmed the proposal of myotoxic sites. It has been observed that these molecules inhibit PLA 2 -like proteins in different ways, including: (i) binding to Helix-I/MDiS region, physically blocking access to the hydrophobic channel (e.g., rosmarinic 48 , 50 , chicoric 30 , and aristolochic acids 4 ); (ii) binding to the MDoS (e.g., caffeic acid 4 and suramin 20 ); (iii) binding in the hydrophobic channel (e.g., caftaric acid 42 , p-bromophenacyl bromide (BPB) 9 ; Varespladib 51 and zinc ions 26 ); (iv) binding to the MDiS (e.g., aristolochic 4 , chicoric acid 30 , zinc ions 26 , suramin 19 , 20 and Varespladib 51 ); or (v) induction of the toxin’s oligomerization (e.g., suramin 19 ). Although the binding regions of the inhibitors are well described, the structural importance of an allosteric activator is only related to the dimer reorientation 14 , 26 .…”

“…The molecular mechanism of these myotoxins remains not yet fully understood, preventing the development of an effective antivenom serum therapy, although recent results based on experimental and theoretical works have shown progress on this area 14 . It is known that PLA 2 -like proteins have a homodimeric assembly which is related to its biological activity 15 , but these myotoxins can present very different quaternary arrangements 14 , 16 – 20 . The homodimeric conformation of PLA 2 -like proteins was further evidenced by the binding of α-tocopherol (αT; vitamin E) to these proteins, which could promote allosteric activation by the dimeric stabilization of the myotoxin BthTX-I from Bothrops jararacussu 17 .…”

Calixarene capture of partially unfolded cytochrome c

“…1). Similar to suramin [28], the 'molecular glue' activity of calixarenes can give rise to interesting protein assembly properties [16][17][18][19][20][21][22][23]. For example, phosphonato-calix [6]arene (pclx 6 ) induces cytc dimers [18], while sulfonato-calix [8]arene (sclx 8 ) gives rise to autoregulated tetramer formation [20].…”