Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions.

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics.