Using Molecular Dynamics Simulations to Assess the Structure and Stability of Transmembrane Oligomeric Intermediates of Pore Forming Proteins

Abstract

Protein membrane interactions play an important role in our understanding of diverse phenomena ranging from membrane-assisted protein aggregation to oligomerization and folding. 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 multimeric transmembrane pore complex through a process of oligomerization. Our understanding of the molecular mechanism of pore formation, specifically for amphiphatic α‑helical toxins is limited. Using molecular dynamics (MD) simulations we examine the process of pore formation of the bacterial α-PFT, cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging form 50- 500 ns, we show that transmembrane oligomeric intermediates or “arcs� form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. We study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the time scale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. The intricate role of cholesterol is elucidated by carrying out all-atom MD simulations (0.6 – 1 microsecond) of a single membrane inserted protomer, a dimer and the dodecameric pore complex. The dual role of cholesterol in assisting the membrane insertion via a cholesterol recognition motif and stabilizing the pore complex is illustrated. We connect the membrane insertion and ensuing conformational change as well as arc formation with recent single molecule and vesicle leakage data from our laboratory.