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

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, alpha(1)- and beta-purothionins (MW: approximately 4.8 kDa), a phosphate ion and a glycerol molecule are modeled bound to the protein. (31)P 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.

Expression, purification and characterization of recombinant crambin.

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues), has been successfully expressed in Escherichia coli from an artificial, synthetic gene. Several expression systems were investigated. Ultimately, crambin was successfully expressed as a fusion protein with the maltose binding protein, which was purified by affinity chromatography. Crambin expressed as a C-terminal domain was then cleaved from the fusion protein with Factor Xa protease and purified. Circular dichroism spectroscopy and amino acid analysis suggested that the purified material was identical to crambin isolated from seed. For positive identification the protein was crystallized from an ethanol-water solution, by a novel method involving the inclusion of phospholipids in the crystallization buffer, and then subjected to crystallographic analysis. Diffraction data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 A at 150 K. The structure, refined to an R value of 9.6%, confirmed that the cloned protein was crambin. The availability of cloned crambin will allow site-specific mutagenesis studies to be performed on the protein known to the highest resolution.

Conversion of lysophospholipids to cyclic lysophosphatidic acid by phospholipase D.

Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine in aqueous 1% Triton X-100 solution. In situ monitoring of this reaction by 31P NMR revealed the formation of cyclic lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an intermediate which was hydrolyzed further by the enzyme at a functionally distinct active site to lysophosphatidic acid (lyso-PA). Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was found to be stable in aqueous neutral solutions at room temperature. It was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate which was approximately 4-fold slower than the rate of formation of cyclic lyso-PA. The addition of 5-10 mM sodium vanadate could partially inhibit the ring opening reaction and thus increase substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act as a dormant configuration of the physiologically active lyso-PA or may even possess specific activities which await verification.