Molecular Dynamics simulations reveal the role of membrane cholesterol in the pore forming pathway of Cytolysin A

Abstract

We investigate the interactions of pore-forming toxins (PFTs) with cholesterol-rich membranes through atomistic molecular dynamics simulations. The PFT of interest is Cytolysin A (ClyA or HlyE). Cytolysin A (ClyA), an pore-forming protein expressed by E. Coli as a water-soluble monomer, undergoes a drastic conformational change from its water-soluble monomeric state to the membrane-inserted protomeric state which subsequently oligomerizes to form a dodecameric pore. Many PFTs have their specificity towards certain receptors, present in the membrane. Recent single molecule experiments show that the conversion rate from the water-soluble monomer to the membrane-inserted protomer increases in the presence of cholesterol. Further, lysis experiments in RBCs have a half-life time that was orders of magnitude smaller than that of RBCs when cholesterol is removed. Consequently, cholesterol was established as factor in enhancing the lytic activity of ClyA. However, the precise 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 s, 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” (a region with high cholesterol affinity) motif and also around residues of the -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 -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. Formations 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 -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. Free energy computations were performed with only the -helix at different orientations in membranes with and without cholesterol. A well defined minima is observed in the presence of cholesterol when compared with the broad minima in the absence of cholesterol. This study has provided molecular level insight into the role of cholesterol in regulating the pore forming activity of ClyA establishing for the first time a specific cholesterol recognizing residues in an -PFT. The identification of receptor specific segments in ClyA could potentially add to the designing of ‘anti-toxin’ therapeutic remedies.