page_title: Lysenin
section_title: Binding, oligomerization and insertion
hierarchical_section_title: Lysenin / Binding, oligomerization and insertion
caption_attribution_description: English: Lysnein molecular mechanism of pore formation.Molecular graphics were performed with the UCSF Chimera package.(Pettersen et al., 2004).
context_page_description: Lysenin is a pore-forming toxin present in the coelomic fluid of the earthworm Eisenia fetida. Pore-forming toxins are a group of proteins that act as virulence factors of several pathogenic bacteria. Lysenin proteins are chiefly involved in the defense against eukaryotic and prokaryotic pathogens. Following the general mechanism of action of PFTs lysenin is segregated as a soluble monomer that binds specifically to a membrane receptor, sphingomyelin in the case of lysenin. After attaching to the membrane, the oligomerization begins, resulting in a nonamer on top of membrane, known as a prepore. After a conformational change, which could be triggered by a decrease of pH, the oligomer is inserted into the membrane in the so-called pore state.
context_section_description: Membrane binding is a requisite to initiate PFT oligomerization. Lysenin monomers bind specifically to sphingomyelin via the receptor binding domain. The final lysenin oligomer is constituted by nine monomers without quantified deviations. When lysenin monomers bind to sphingomyelin-enriched membrane regions, they provide a stable platform with a high lateral mobility, hence favouring the oligomerization. As with most PFTs, lysenin oligomerization occurs in a two-step process, as was recently imaged.
  The process begins with monomers being adsorbed into the membrane by specific interactions, resulting in an increased concentration of monomers. This increase is promoted by the small area where the membrane receptor accumulates owing to the fact that the majority of PFT membrane receptors are associated with lipid rafts. Another side effect, aside from the increase of monomer concentration, is the monomer-monomer interaction. This interaction increases lysenin oligomerization. After a critical threshold concentration is reached, several oligomers are formed simultaneously, although sometimes these are incomplete. In contrast to PFTs of the cholesterol-dependent cytolysin family, the transition from incomplete lysenin oligomers to complete oligomers has not been observed.
  A complete oligomerization results in the so-called prepore state, a structure on the membrane. Determining the prepore's structure by X-ray or Cryo-EM is a challenging process that so far has not produced any results. The only available information about the prepore structure was provided by Atomic Force Microscopy (AFM). The measured prepore height was 90 Å; and the width 118 Å, with an inner pore of 50 Å. A model of the prepore was built aligning the monomer structure (PDB: 3ZXD​) with the pore structure (PDB: 5GAQ​) by their receptor-binding domains (residues 160 to 297). A recent study in aerolysin suggests that the currently accepted model for the lysenin prepore should be revisited, according to the new available data on the aerolysin insertion.
  A conformational change transforms the PFM into the transmembrane β-barrel, leading to the pore state. The trigger mechanism for the prepore-to-pore transition in lysenin depends on three glutamic acid residues (E92, E94 and E97), and is activated by a decrease in pH, from physiological conditions to the acidic conditions reached after endocytosis, or an increase in calcium extracellular concentration. These three glutamic acids are located in an α-helix that forms part of the PFM, and glutamic acids are found in aerolysin family members in its PFMs. Such a conformational change produces a decrease in the oligomer height of 2.5 nm according to AFM measurements. The main dimensions, using lysenin pore X-ray structure, are height 97 Å, width 115 Å and the inner pore of 30 Å. However, complete oligomerization into the nonamer is not a requisite for the insertion, since incomplete oligomers in the pore state can be found. The prepore to pore transition can be blocked in crowded conditions, a mechanism that could be general to all β-PFTs. The first hint of crowding effect on prepore to pore transition was given by congestion effects in electrophysiology experiments. High-Speed AFM studies incubating lysenin on sphingomyelin/cholesterol membranes has shown that under crowded conditions the prepore to pore transition gets blocked by steric interactions.
caption: Lysenin mechanism of action Scheme. a) Lysenin monomers are segregated as soluble proteins that bind specifically to sphingomyelin by its receptor binding domain. After binding, and reach a certain density, the oligomerization starts. b) After a complete oligomerization, the prepore is formed. The prepore model shown here was assembled from the monomer structure and aligned with the pore structure (PDB: 5GAQ​) by their receptor-binding domains (residues 160 to 297). The height of the prepore was set to agree with the Atomic Force Microscopy measurements. c) Membrane inserted Lysenin assembly (PDB: 5GAQ​). The height of the pore was measured from the detergent belt to the last residue, assuming that the detergent belt corresponds with the part of the pore surrounded by the membrane. The membrane was placed in the β-barrel of the pore to match with the detergent belt, that englobe all the hydrophobic residues of the β-barrel. The hydrophobic surface colour scale is according to the hydrophobicity scale of Kyte and Doolittle.
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