J Pharm Pharmacogn Res 3(suppl. 1): S90, 2015

Proceedings of the 4th International Symposium on Pharmacology of Natural Products FAPRONATURA 2015  September 21st-25th, 2015; Cuban Society of Pharmacology. Topes de Collantes, Sancti Spiritus, Cuba.

Oral Communication


Mesa-Galloso H1, Delgado-Magnero KH1, Pedrera L1, Hernández-González JE1, Cabezas S1, Alvarez C1, Lanio ME1, García-Saez A2, Ros U1, Valiente PA1.

1Center for Protein Studies, Faculty of Biology, Havana University, Cuba. E-mail: haydee@fbio.uh.cu
2Interfaculty Institute for Biochemistry, University of Tubingen, Tubingen, Germany.


Introduction: Actinoporins are soluble pore-forming isolate from sea anemones. The biomedical application under development of this toxin has been devised as inmunotoxins against undesirable cells and toxin-liposome complexes to deliver molecules to cell cytosol. The three-dimensional (3D) structure of equinatoxin II (EqtII), sticholysins I and II (StI, and StII), and fragaceatoxin C (FraC) have been solved. The mechanism of pore-formation of actinoporins is based on an initial binding step followed by membrane insertion of the N-terminal sequence and oligomerization. Recently, was proposed an octameric 3D pore structure for FraC formed via protein dimerization and partial unfolding of the N-terminal region, suggesting similar pore architecture for the others eukaryotic actinoporins. However, mutagenesis experiments to clarify if the oligomerization interface is conserved among the actinoporins family are still lacking. Methods: Here, we designed a double mutant to disrupt the putative actinoporins oligomerization interface by combining sequence and structure analysis with free energy calculations, and coarse grained (CG) molecular dynamics (MD) simulations. Results: We observed that the presence of both mutations at positions V60 and F163, decreased the stability of the 3D oligomeric structure by disrupting the structure complementarities of this hydrophobic region, and impairing dimer formation. A free energy calculation supports our hypothesis and predicts the electrostatic repulsion among V60D of one protomer face and F163D, and E173 in another protomer face as the driving force to destabilize the oligomerization step. To test our predictions, we obtained, expressed and purified the double mutants FraCV60D/F163D, EqtIIV60D/F163D, and StIII58D/I161D. Circular dicroism and fluorescent spectroscopy showed a similar spectrum for the three mutants compared to the native proteins. Finally, the functional characterization by fluorescence and hemolytical activity techniques revealed that mutants kept a similar ability to bind model membranes while loss completely their pore-forming activity. Conclusions: Our results indicate that actinoporins share a conserved oligomerization interface.

Citation Format: Mesa-Galloso H, Delgado-Magnero KH, Pedrera L, Hernández-González JE, Cabezas S, Alvarez C, Lanio ME, García-Saez A, Ros U, Valiente PA (2015) Actinoporins, pore-forming proteins from sea anemones, their oligomerization pathway. [Abstract]. In: Proceedings of the FAPRONATURA 2015; 2015 Sep 21-25; Topes de Collantes, Sancti Spiritus: CSF. J Pharm Pharmacogn Res 3(Suppl. 1): S90. Abstract nr OC-78.