Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein

Abstract

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel.

Electron cryomicroscopy as a powerful tool in biomedical research

“…4b) [86]. The cryo-EM structures of a number of pore-forming toxins that assemble into large pores to disturb essential cellular gradients, usually featuring > 10 subunits, were also determined, giving important insights into conformational changes during membrane insertion [87, 88]. Cryo-EM studies on Tc toxin complexes from Photorhabdus luminescens revealed a unique syringe-like injection and translocation mechanism for membrane permeation and delivery of the toxic component into host cells (Fig.…”

Advances in structure determination by cryo‐EM to unravel membrane‐spanning pore formation

“…Lysenin is a component of the earthworm's immune system, exerting its function by attacking membranes of parasites . In addition to the X‐ray structure of the lysenin monomer, the 3.1 Å structures of the pore, determined independently using cryo‐EM and X‐ray crystallography, have helped to identify the functional folds and interactions of the three separate domains of the lysenin monomer [Fig. (A,B)].…”

“…In contrast, the cap‐domain exhibits a moderate structural deformation while the PFM undergoes a major conformational alteration, with some residues moving as far as 100 Å, during prepore‐to‐pore formation. The crystal structure of lysenin in complex with sphingomyelin combined with a bending deformation of the cap domain further suggests that structural changes triggered in the cap domain might include the molecular sensor that initiates the prepore‐to‐pore transition. These structural data have been instrumental in designing experiments to better understand lysenin pore formation versus substrate interaction events .…”

“…, blue dots in magenta boxes) that facilitate the formation of the β‐barrel pore. Morphing analyses between several prepore and pore states suggests that β‐PFP maturation involves a swirling membrane insertion mechanism of the PFM …”

“…The cryo‐EM structure revealed that in the lysenin pore state, the positioning of this binding pocket is ideally situated for interactions with PC or SM in the membrane [Fig. (B)] . Both intrachain and interchain contacts are primarily conserved throughout the receptor‐binding domain of the lysenin nonamer in both the prepore and the pore states [Fig.…”