(712i) String Method Based Free Energy Calculations Reveal the Role of Membrane Cholesterol in Bacterial Toxin Activity

Pore-forming toxins are a class of proteins expressed by bacteria as water-soluble monomers that convert to membrane-embedded protomers upon interaction with the plasma membrane. Subsequently, protomers oligomerize in the membrane to form transmembrane pores. Unregulated pore formation causes ion imbalance and passage to other pathogens, leading to cell death and various bacterial infections. With rising bacteria resistance, understanding the molecular mechanisms of pore formation could lead to alternate treatment protocols. Cytolysin A (ClyA), a 34 kDa pore-forming toxin expressed by E. coli, undergoes large conformational changes upon pore formation. Previously, cholesterol has been shown to increase the activity of ClyA by stabilizing the pore structure in the membrane. However, it is unclear if cholesterol plays a significant role in the conformational changes that occur during the membrane-assisted monomer to protomer transition of the protein. One major highlight of the conformational change is the conversion of the β-tongue from a β-sheet to a helix-turn-helix motif in the membrane. In this work, we performed all-atom molecular dynamics simulations of the β-tongue embedded in the membrane with and without cholesterol and utilized the string method approach combined with path collective variables to estimate the free energy landscape along the transition path. The free energy landscape reveals the formation of an intermediate unfolded state in the membrane containing cholesterol; however, the tendency to unfold becomes less probable in the absence of cholesterol. Thus, cholesterol is found to promote the formation of an unfolded intermediate state in the conversion of the β-sheet to a helix-turn-helix motif during pore formation. Free energy analysis shows a higher free energy barrier and the formation of a helix-turn-helix motif of the protomer state with lowered stability in the absence of cholesterol. Combined with our earlier findings, our free energy computations reveal that cholesterol plays a wider and more complex role in the pore-forming pathway of ClyA. In addition to stabilizing the pore state, our analysis indicates that cholesterol increases the kinetics of the conformational change and stabilizes the protomer state in the membrane. Cholesterol might also play a similar role in the pore-forming pathway of other cholesterol-dependent pore-forming toxins such as Listeriolysin O and Pneumolysin.