Autocatalytic Acylation of Phospholipase-like Myotoxins

Abstract

Several snake venoms contain a phospholipase A2 in which position 49 in the active site is occupied by a lysine or a serine instead of the aspartate residue normally found. Although these proteins do not bind Ca2+ and are devoid of catalytic activity, they are still highly specific myotoxins and have recently been shown to induce membrane leakage by a new type of cytolytic mechanism. Three of these toxins, myotoxin II from Bothrops asper, ammodytin L from Vipera ammodytes, and the K49 protein from Agkistrodon piscivorus piscivorus, were examined for their interaction with fatty acids and were found to bind long-chain fatty acids covalently by a rapid, spontaneous, autocatalytic process. The fatty acids could be released by treatment with 1 M NH2OH or NaOH, but not with 1 M NaCl or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron spin resonance studies using spin-labeled fatty acids showed that only the carboxyl headgroup of the fatty acid was linked to protein amino acid, the carbon chain had free mobility and did not bind tightly to the protein surface. Stearic acid methyl esters and short-chain fatty acids did not bind to the toxins. Acylated myotoxins bound to the surface of liposomes and isolated muscle membranes, with the fatty acid moiety inserted into the lipid bilayer and possibly acting as an anchor. The phospholipase-like myotoxins represent the first group of proteins able to undergo acylation by spontaneous reaction with free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in bothropstoxin I, a dimeric Lys49-phospholipase A2 homologue

“…This high stability in solution suggests that the dimer may be of biological relevance and that the ''open'' to ''closed'' transition might be of significance for the Ca 2ϩ -independent membrane damaging activity observed in Lys49-PLA2 homologues. 23,24 The MT-II from B. asper has been shown to covalently bind lipid substrates, which may be a consequence of an ''interrupted'' catalytic cycle 51 and it has been proposed that this modification serves to anchor the protein to the lipid bilayer. Furthermore, a synthetic peptide of the C-terminal region of the MT-II, which is fully conserved in the BthTX-I, reproduces at reduced levels the cytolytic activity of the intact protein, 52 indicating a role of this region in the disruption of membrane integrity.…”

Snake Venom Phospholipases A2: A Novel Tool Against Bacterial Diseases

“…Identical interactions are observed in myotoxins as a consequence of a coupled conformational change involving Gly 30 and neighboring residues of the Ca +2 -binding loop and residues 115-125 [41]. The buried conformation adopted by Lys 122 in the ligand-bound form leads to increased hydrogen bonding between the C-terminal region (residues [115][116][117][118][119][120][121][122][123][124][125] 54], and the potential acylation of these toxins via autocatalysis [55], or via an interrupted catalytic cycle that fails to release a free fatty acid [56] which may enhance membrane perturbation. This would be the key toxic event, allowing an uncontrolled influx of ions (i.e.…”

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

“…Notwithstanding this observation, there now appears to be ample evidence that this family of myotoxins is able to bind fatty acid and possibly other long chain ligands. Electron spin resonance experiments (77) and the crystal structures of Lys 49 -PLA2s, in which fatty acid molecules have been fortuitously observed within the hydrophobic channel (45,47), have until recently been the most convincing sources of evidence. Recently, however, the crystal structure of Bothrops asper myotoxin II, in which stearic acid was clearly observed in the electron density maps bound to the protein after co-crystallization, seems to provide the final word.…”