During a pandemic, research for efficient and specific therapeutics against severe illnesses attracts attention from publicity world-wide. Especially antibiotic resistances jeopardize people in third-world countries with limited public health infrastructure. Understandably, diseases such as sepsis, meningitis or pneumonia caused by Streptococcus Pneumonia infection have a deadly potential in those areas.
One of the main factors involved in S.Pneumonia action is Pneumolysin (PLY), a cholesterol-dependent cytolysin (CDC). Water-soluble CDCs are secreted, oligomerize on cholesterol-rich membranes into pre-pores and transform into large pores, thereby lysing the target cell. During the pre-pore-to-pore transition, the protomers undergo a huge conformational change refolding alpha-helices into transmembrane beta-sheets.
Hitherto, structural information was missing to understand the molecular interactions during this process to ease therapeutic drug research. Understanding the whole pore-forming mechanism would allow to develop drugs that interfere with pore formation. To obtain high-resolution structures of CDCs, cryo-electron microscopy is the method of choice, because it allows the structure determination of protein complexes of several MDa in size in a close-to-native environment. However, the size heterogeneity of the pre-pore or pore complexes is challenging. For wildtype PLY, stabilization of the pore complexes by amphipols was a key step and resulted in a structure resolved to 4.5 Å . We have refined this method and have improved the resolution of the pore complex to ~3.5 Å, which is high enough to assign the sidechains with high accuracy. The pre-pore complex could not be stabilized similarly, because pre-pores are attached to the intact membrane surface. Introducing mutations to lock the pre-pore complex on cholesterol-rich liposomes has been the solution to get a higher-resolution structure at ~4.5 Å.
The presented structures constitute a fundament in scope of research of new and high-efficient therapeutic drugs against diseases caused by PLY and other CDCs predicted to undergo the same pore-forming mechanism.