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Molecular mechanisms of lipid membrane disruption induced by pore forming proteins

Pore-forming proteins (PFPs) disrupt the normal functions of biological membranes by affecting their integrity and selective permeability. They are expressed by organisms of all kingdoms of life. In most cases they play a role in attack or defence, and thus act as toxins; however, in some organisms they have also been found in immune response, development or digestion. PFPs are expressed as water-soluble monomers that bind specifically or nonspecifically to either lipid, sugar or protein components of target membranes. Membrane-bound monomers then fuse to form structured oligomers, followed by coordinated conformational changes of all protomers, resulting in the formation of functional transmembrane pores. The transmembrane channels of these pores, are lined by either symmetric α-helical clusters or β-barrels, based on which PFPs are classified as α- or β-PFPs.

We are interested in the molecular mechanisms of membrane disintegration by PFPs. We study the molecular interactions of PFPs with lipid membranes and search for specific receptors or enhancers that affect their pore-forming activity, using various biochemical and biophysical approaches. In addition, we structurally analyse both soluble monomers and transmembrane pores using X-ray crystallography and cryo-transmission electron microscopy.

We determined the crystal structure of the pore formed by lysenin, a member of the aerolysin family of PFPs from earthworm, and described its mechanism of assembly into compact pores in sphingomyelin-rich membranes (Podobnik et al., 2016). In addition to biological relevance, this study also demonstrated the potential of these pores for application in nanobiotechnology. We exploited the natural pH-dependent mechanism of pore formation by listeriolysin O (LLO), a cholesterol dependent cytolysin (CDC), and further engineered this protein, resulting in a strictly pH-regulated mutant (Kisovec et al., 2017). We produced LLO in giant vesicles (GUVs) made of archaeal lipids by cell-free expression, which could be beneficial for applications in synthetic biology (Rezelj et al., 2018). We are also interested in the evolution of a pore-forming domain of PFPs using methods of in vitro evolution of lipid-binding sites via ribosomal display, which was used to search for specific protein inhibitors of PFPs (Omersa et al., 2020). The same method revealed atypical variants of the membrane-binding domain of another CDC, perfringolysin O (Šakanović et al., 2020). We were the first to discover the glycolipid receptors (GIPCs) of actinoporin-like Nep1-like proteins (NLPs). NLPs are expressed by oomycetes, fungi and bacteria, and are toxic to (economically relevant) plants; we described their mechanism of action, characterized major structural differences between toxic and non-toxic members, and proposed lead molecules for potential inhibition of these plant toxins (Lenarčič et al., 2017, 2019; Pirc et al., 2021, 2022). We hae also determined the crystal structure of aegerolysin from the human pathogen Pseudomonas aeruginosa and its interaction with the membrane receptor ceramide phospho-ethanolamine (Kočar et al., 2017), and demonstrated that a fungus, Beauveria bassiana, which is an active ingredient of bioinsecticides, expresses an aegerolysin BlyA that also interacts with an insect-specific membrane lipid ceramide phosphoethanolamine (Kraševec et al., 2021).

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