Molecular Dynamics Simulations Reveal the Role of Membrane Cholesterol during Pore Forming Pathway of Cytolysin A

Abstract

Cytolysin A (ClyA), an α pore-forming toxin expressed by E. Coli as a water-soluble monomer, undergoes a large conformational change during pore formation. Although cholesterol is known to enhance the lytic activity of ClyA, molecular aspects of the interactions between cholesterol and ClyA during pore formation are not well understood. Using all-atom molecular dynamics simulations ranging from 0.5 - 0.9 µs, we study a single membrane-inserted protomer, a dimer (two protomers) and the dodecameric ClyA pore embedded in a DOPC+30% cholesterol bilayer. In the single membrane-inserted protomer, high cholesterol occupancy was observed around the transmembrane residues of N-terminus which form part of a CRAC motif and also around residues of the β-tongue. Although high cholesterol occupancy sites were not observed near the N-terminus in the dimer simulations, a cholesterol molecule was preferentially located in the pocket formed between two adjacent β-tongues of the dimer. Cholesterol spent 97% of the simulation time (600 ns) inside this pocket sampling two major orientations. Energies of two conformations were reported from docking simulations. Formation of transmembrane water channels were observed in both single membrane inserted and dimer ClyA simulations. From the dodecameric pore simulations, density map showed regions of high cholesterol population between the β-tongue pockets and mobility map indicated slower cholesterol in the vicinity of the pore as compared to bulk. Our simulations elucidate specific interactions with cholesterol that could stabilize both the single membrane inserted protomeric state as well as the dodecameric pore.