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J Biol Chem. 2016 Sep 9;291(37):19210-19219. doi: 10.1074/jbc.M116.734053. Epub 2016 Jul 21.

Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins.

Author information

1
From the Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan.
2
the Department of Biochemistry and Molecular Biology and.
3
Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain.
4
the Structural Biology Unit, Center for Cooperative Research in Biosciences, CICbiogune, 48160 Derio, Spain.
5
the U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France, and.
6
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.
7
the Institute of Medical Science, University of Tokyo, Minato-ku, 108-8639 Tokyo, Japan.
8
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.

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.

KEYWORDS:

atomic force microscopy (AFM); cryo-electron microscopy; cytolysin; lipid vesicle; lipid-protein interaction; oligomerization; pore forming protein; protein structure

PMID:
27445331
PMCID:
PMC5016661
DOI:
10.1074/jbc.M116.734053
[Indexed for MEDLINE]
Free PMC Article

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