Proposal for molecular mechanism of thionins deduced from physico-chemical studies of plant toxins

• Published in: The journal of peptide research Vol. 64; no. 6; pp. 210 - 224
• Main Authors: Stec, B., Markman, O., Rao, U., Heffron, G., Henderson, S., Vernon, L.P., Brumfeld, V., Teeter, M.M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.12.2004
• Subjects: Antimicrobial Cationic Peptides; Binding Sites; Cell Membrane - chemistry; Cell Membrane - metabolism; Crystallography, X-Ray; Fluorescence Polarization; Glycerophosphates - chemistry; Magnetic Resonance Spectroscopy; mechanism of toxicity; membrane lysis; Models, Chemical; Models, Molecular; Phospholipids - chemistry; Phospholipids - metabolism; Plant Proteins - chemistry; Plant Proteins - metabolism; plant toxins; Pyrularia - chemistry; Sequence Alignment; Solubility; thionins; Toxins, Biological - chemistry; Toxins, Biological - metabolism
• ISSN: 1397-002X; 1399-3011
• DOI: 10.1111/j.1399-3011.2004.00187.x

:  We propose a molecular model for phospholipid membrane lysis by the ubiquitous plant toxins called thionins. Membrane lysis constitutes the first major effect exerted by these toxins that initiates a cascade of cytoplasmic events leading to cell death. X‐ray crystallography, solution nuclear magnetic resonance (NMR) studies, small angle X‐ray scattering and fluorescence spectroscopy provide evidence for the mechanism of membrane lysis. In the crystal structures of two thionins in the family, α1‐ and β‐purothionins (MW: approximately 4.8 kDa), a phosphate ion and a glycerol molecule are modeled bound to the protein. 31P NMR experiments on the desalted toxins confirm phosphate‐ion binding in solution. Evidence also comes from phospholipid partition experiments with radiolabeled toxins and with fluorescent phospholipids. This data permit a model of the phospholipid–protein complex to be built. Further, NMR experiments, one‐dimensional (1D)‐ and two‐dimensional (2D)‐total correlation spectroscopy (TOCSY), carried out on the model compounds glycerol‐3‐phosphate (G3P) and short chain phospholipids, supported the predicted mode of phospholipid binding. The toxins’ high positive charge, which renders them extremely soluble (>300 mg/mL), and the phospholipid‐binding specificity suggest the toxin–membrane interaction is mediated by binding to patches of negatively charged phospholipids [phosphatidic acid (PA) or phosphatidyl serine (PS)] and their subsequent withdrawal. The formation of proteolipid complexes causes solubilization of the membrane and its lysis. The model suggests that the oligomerization may play a role in toxin's activation process and provides insight into the structural principles of protein–membrane interactions.