Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A

Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the β-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded β-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 μs of all-atom thermal unfolding MD simulations of wild-type ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the β-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the β-tongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the β-tongue as well as N-terminal mutants that lower the tendency to unfold and disorder the β-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the β-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the β-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.