Studying Binding, Conformational Transition and Assembly of E. Coli Cytolysin a Pore Forming Toxin by Single Molecule Fluorescence

Pore forming toxins (PFT) belong to a class of bacterial toxin proteins that form nano-scaled pores on target cell's membrane and cause unregulated efflux of ions and biomolecules leading to cell death. They are released in a water-soluble conformation which upon membrane exposure undergoes large structural rearrangement. This membrane bound monomer further oligomerizes and forms of a complete pore. While high resolution structural information of complete pores are available, our understanding of early events of PFT binding and assembly is incomplete owing to the highly dynamic nature of the aforementioned processes. In this study, we use single molecule tracking and spectroscopy to understand the dynamics of Cytolysin A (ClyA), a prototypical α-PFT from E. coli, on lipid bilayer membranes. Binding of ClyA to PEG-cushioned supported bilayer was rapid and reached saturation within a few seconds. Diffusional analysis of particle trajectories showed existence of two discrete mobility states exhibiting ‘fast’ and ‘slow’ motions. Binding of PFT proteins was invariably in the ‘fast’ mobility state that was followed by transitions of the single monomer between the two states. The slow moving population was significantly enhanced in cholesterol containing bilayers. We argue that the change in mobility is a consequence of structural transition to an assembly competent intermediate, the protomer state. Preliminary analysis indicates that cholesterol enhances conformational transition by direct binding to the N-terminus of ClyA leading to stabilization of the protomer-like state. This stabilization directly translates to increased formation of higher order structures at high concentrations of protein as measured by analysis of single molecule photobleaching trajectories of pre-formed ClyA pores. Therefore, we propose a molecular mechanism for selective pore formation in eukaryotic membranes driven by conformational selectivity in the presence of cholesterol.