Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha(α)-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the β-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for β-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the β-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the β-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the β-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the β-tongue and the tendency to induce disorder in the β-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general.
Anchoring Intrinsically Disordered Proteins to Multiple Targets: Lessons from N-Terminus of the p53 Protein