Utilizing an aqueous-liquid crystal interface to investigate membrane protein interactions and mutation effects of a pore-forming toxin

Listeriolysin O (LLO) is a crucial cholesterol-dependent cytolysin (CDC) secreted by <i>Listeria monocytogenes</i>. LLO lyses the phagosomal membrane <i>via</i> pore-formation, resulting in pathogenesis. CDCs' ability to recognize and bind to membrane cholesterol is a hallmark in the pathogenesis of these pore-forming toxins, distinguishing them from other toxins. Conservation of the cholesterol-recognition motif (CRM) has been discovered to be one of the prerequisites for the membrane binding of some CDCs, but the role of the CRM for LLO binding and pore-formation is still unclear. Therefore, we investigated LLO-mediated lipid remodelling at a nanomolar concentration using the interfacial properties of a biomimetic liquid crystal (LC)-aqueous interface. The examination addresses the significance of the CRM in protein structure and membrane reorganizations for the cholesterol-mediated binding of LLO. We report that the CRM assists in the binding of LLO in a unique amphipathic environment, especially at low cholesterol levels. However, eliminating or substituting the CRM from LLO significantly alters the threshold cholesterol level required for its activity. This study also reveals the effect of cholesterol-dependent membrane dynamics in the association and activity of LLO. Our findings suggest a novel paradigm that opens up an array of possibilities for discovering sequential mutations and delineating the molecular mechanisms of CDCs in nanomolar concentration regimes.