title: Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins.
abstract: Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.
authors.affiliation: From the Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan.|the Department of Biochemistry and Molecular Biology and.|Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain.
authors.forename: Koldo
authors.identifier: 0000-0002-9877-9168
authors.initials: K
authors.lastname: Morante
authors.affiliation: the Department of Biochemistry and Molecular Biology and.|Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain.
authors.forename: Augusto
authors.initials: A
authors.lastname: Bellomio
authors.affiliation: the Structural Biology Unit, Center for Cooperative Research in Biosciences, CICbiogune, 48160 Derio, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Gil-Cartón
authors.affiliation: the U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France, and.
authors.forename: Lorena
authors.initials: L
authors.lastname: Redondo-Morata
authors.affiliation: the Department of Biochemistry and Molecular Biology and.|Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Sot
authors.affiliation: the U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France, and.
authors.forename: Simon
authors.initials: S
authors.lastname: Scheuring
authors.affiliation: the Structural Biology Unit, Center for Cooperative Research in Biosciences, CICbiogune, 48160 Derio, Spain.
authors.forename: Mikel
authors.initials: M
authors.lastname: Valle
authors.affiliation: the Department of Biochemistry and Molecular Biology and.
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: González-Mañas
authors.affiliation: From the Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan, tsumoto@bioeng.t.u-tokyo.ac.jp.|the Institute of Medical Science, University of Tokyo, Minato-ku, 108-8639 Tokyo, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.affiliation: From the Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan, jose@bioeng.t.u-tokyo.ac.jp.
authors.forename: Jose M M
authors.identifier: 0000-0001-5568-2369
authors.initials: JMM
authors.lastname: Caaveiro
mesh_terms: D000818:Animals; D003064:Cnidarian Venoms / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry*; D012615:Sea Anemones / Q000737:chemistry*
chemical_list: D003064:Cnidarian Venoms; D008567:Membranes, Artificial; D052899:Pore Forming Cytotoxic Proteins; C545670:fragaceatoxin C
keywords: atomic force microscopy (AFM); cryo-electron microscopy; cytolysin; lipid vesicle; lipid-protein interaction; oligomerization; pore forming protein; protein structure