Even though the structures of PLYD168A and PLYΔ146/147 were very similar to PLYWT, they displayed major differences in their membrane-binding and oligomerization behaviour. Like PLYWT, PLYD168A bound to cholesterol-containing liposomes and oligomerized into rings (Figure 7A). However, unlike PLYWT that lysed the liposomes within a short time, liposomes incubated with PLYD168A remained intact for hours. Frequently, the rings detached from the liposomes, indicating they were prepores that had not yet inserted into the membrane. This is also reflected by the hemolytic activity, which was reduced by 80% compared to PLYWT, highlighting the important role of Asp168 in ionic interactions between β-strands that stabilize the pore complex.