Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

Abstract

Lys49-phospholipases A₂ (Lys49-PLA₂s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA₂s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA₂ from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA₂s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA₂s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA₂s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA₂s.

Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids

“…close to 1.0 Å ( Table 2 ). These values are comparable to other Lys49-PLA 2 s [ 35 , 48 ], where a pattern was observed among apo forms with an r.m.s.d. of approximately 1.0 Å, while for structures that present with molecules in the hydrophobic channel (e.g., PEG and α-tocopherol)these values are significantly lower.…”

“…PrTX-I/AA,PrTX-I/CA and PrTX-I/RA have 40°,39° and 43° torsion angles and 27°, 28° and 23°aperture angles, respectively. These values are in agreement with structures in the active state [ 35 , 48 ]. In contrast, inactive (or apo) structures have higher values of torsion angles (60°-61°) and smaller values of aperture angles (6°-7°) [ 35 , 48 ].…”

PLA2-like proteins myotoxic mechanism: a dynamic model description

“…The disturbance of membranes by PLA 2 -like proteins has been associated with C-terminal residues 9 , 10 and is enhanced if the quaternary structure is a homodimeric assembly 11 , 12 . Under physiological conditions, dimers are observed by dynamic light scattering 13 – 15 , non-reducing SDS-PAGE results 16 – 20 , and SAXS experiments 21 . Two different dimers have been proposed according to crystallography experiments: a large dimer with an interface composed of β-wings and a compact dimer with an interface composed of calcium binding loops.…”

Cellular Mechanisms of Action of Snake Phospholipase A2 Toxins

“…The resolution of a higher number of PLA2 structures, together with a comparison of the amino acid Snake Venoms DOI 10.1007/978-94-007-6648-8_26-1 # Springer Science+Business Media Dordrecht 2015 lateral chains exposed in 3D models, will allow to identify 3D motifs crucial for a defined pharmacological activity. Some recent papers are moving in this direction (Dos Santos et al 2009;Cendron et al 2012;Faure and Saul 2012;Salvador et al 2013). Figure 2 reports a statistical analysis of snake PLA2s presently annotated by the UniProtKB/Swiss-Prot Tox-Prot program.…”