Assessing the Structure of Transmembrane Oligomeric Intermediates of an α‑Helical Toxin using Molecular Dynamics Simulations

Pore forming toxins (PFTs) are the primary vehicle for infection by several strains of bacteria. These proteins which are expressed in a water soluble form (monomers), bind to the target membrane and conformationally transform (protomers), and self-assemble to form a multimer transmembrane pore complex through a process of oligomerization. PFTs are broadly classified into or alpha or beta toxins. In contrast to beta-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains have hindered our understanding of alpha-PFT pore formation. We use molecular dynamics (MD) simulations to examine the process of pore formation of the bacterial toxin Cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging from 50- 500 ns, we show that transmembrane oligomeric intermediates or ‘arcs’ form stable proteo-lipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the timescale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella sampling with a coarse grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane. A single inserted protomer has a stabilization free energy of 53 kJ/mol and forms a stable transmembrane water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore formation pathway for ClyA.