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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 ?-PFTs. However, in the class of ?-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 ?-PFT.

SUBMITTER: Morante K

PROVIDER: S-EPMC5016661 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Morante Koldo K Bellomio Augusto A Gil-Cartón David D Redondo-Morata Lorena L Sot Jesús J Scheuring Simon S Valle Mikel M González-Mañas Juan Manuel JM Tsumoto Kouhei K Caaveiro Jose M M JMM

The Journal of biological chemistry 20160721 37

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 produ ...[more]

PMID: 27445331

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Project description:Membrane-bound transcription factors (MTFs) are located in cellular membranes due to their transmembrane domains. In plants, proteolytic processing is considered to be the main mechanism for MTF activation, which ensures the liberation of MTFs from membranes and further their translocation into the nucleus to regulate gene expression; this process skips both the transcriptional and translational stages, and thus it guarantees the prompt responses of plants to various stimuli. Currently, information concerning plant MTFs is limited to model organisms, including Arabidopsis thaliana and Oryza sativa, and little is known in other plant species at the genome level. In the present study, seven membrane topology predictors widely used by the research community were employed to establish a reliable workflow for MTF identification. Genome-wide in silico analysis of MTFs was then performed in 14 plant species spanning the chlorophytes, bryophytes, gymnosperms, monocots and eudicots. A total of 1,089 MTFs have been identified from a total of 25,850 transcription factors in these 14 plant species. These MTFs belong to 52 gene family, and the top six most abundant families are the NAC (128), SBP (77), C2H2 (70), bZIP (67), MYB-related (65) and bHLH (63) families. The MTFs have transmembrane spans ranging from one to thirteen, and 71.5% and 21.1% of the MTFs have one and two transmembrane motifs, respectively. Most of the MTFs in this study have transmembrane motifs located in either N- or C-terminal regions, indicating that proteolytic cleavage could be a conserved mechanism for MTF activation. Additionally, approximately half of the MTFs in the genome of either Arabidopsis thaliana or Gossypium raimondii could be potentially regulated by alternative splicing, indicating that alternative splicing is another conserved activation mechanism for MTFs. The present study performed systematic analyses of MTFs in plant lineages at the genome level, and provides invaluable information for the research community.

Project description:The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-β-D-glucoside micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than a toxin monomer in membrane perturbation of calcein-loaded liposomes. A three-dimensional model of toxin trimer obtained by negative-stain EM in combination with single-particle reconstruction at ∼5 nm resolution showed a propeller-shaped structure with 3-fold symmetry. Fitting the three-dimensional reconstructed EM map with a 100-ns molecular dynamics-simulated Cry4Ba structure interacting with an octyl-β-D-glucoside micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy imaging at nanometer resolution and a subsecond frame rate demonstrated conformational transitions from a propeller-like to a globularly shaped trimer upon lipid membrane interactions, implying prepore-to-pore conversion. Real-time trimeric arrangement of monomers associated with L-α-dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid bicelle membranes was also envisaged by successive high-speed atomic force microscopy imaging, depicting interactions among three individual subunits toward trimer formation. Together, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for the molecular assembly of a prepore trimer capable of inserting into target membranes to generate a lytic pore.

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

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

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