Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes

Abstract

Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.

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

“…For the cardiotoxins as is suggested here for thionins, a hydrophobic surface of the molecule was implicated in membrane binding. The evidence pointed toward the involvement of a Trp residue (12) in initiation of the membrane binding. It is also known that cardiotoxins interact with membranes directly by binding to a patch of negatively charged phospholipids (44,49).…”

“…The identity of the tightly bound lipid was difficult to determine, but it was previously suggested to be a derivative of diacyl‐glycerol. More recent studies of Pyrularia toxin implicated negatively charged phospholipids such as phosphatidic acid (PA) and phosphatidyl serine (PS) (12).…”

“…There is a significant groove between these two segments. The sequence alignment of diverse thionins indicates that the five amino acids (9)(10)(11)(12)(13)(14) that line the cleft are the most strongly conserved fragment of the molecule besides the structural cysteine residues ( Fig. 1).…”

Plant thionins – the structural perspective

“…The identity of the tightly bound lipid has not always been determined, but it was indicated to be a derivative of diacyl-glycerol. More recent studies of Pyrularia toxin implicated negatively charged phospholipids, such as phosphatidic acid and phosphatidyl serine [35], but studies with artificial membranes showed that thionins most likely interact with a much wider class of phospholipids [36]. There are several independently proposed functions for these small cysteine-rich basic peptides.…”

Interaction of Viscotoxins A3 and B with Membrane Model Systems: Implications to Their Mechanism of Action

“…Studies of thionin toxicity suggest the requirement for a electrostatic interaction of the positively charged proteins with the negatively charged phospholipids of the membrane leading to major changes in the structure and integrity of the membrane as have been observed in both cell plasma membranes and model membranes (Caaveiro et al, 1998;Huang et al, 1997;Hughes et al, 2000;Wang et al, 1993;Wilson et al, 1997). It is unclear why amphipathic polypeptides such as thionins from mistletoe and other plants sharing a great extent of structural identity show quite different biological behavior.…”