Assessing the Structure and Stability of Transmembrane Oligomeric Intermediates of an α-Helical Toxin

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 multimer transmembrane pore complex through a process of oligomerization. On the basis of the structure of the transmembrane domains, PFTs are broadly classified into β or α toxins. In contrast to β-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains has hindered our understanding of α-PFT pore formation. In this article, we use molecular dynamics (MD) simulations to 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 from 50 to 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. 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 time scale 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 -52.9 ± 1.2 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.

Transmembrane oligomeric intermediates of pore forming toxin Cytolysin A determine leakage kinetics

“…In recent molecular dynamics simulations of ClyA, carried out in our laboratory we have shown that membrane inserted arcs or pores are lined by the protein on one face with toroidal lipids forming the pore interior in regions where proteins are absent. 31 These arcs were found to be stable structures with a water channel capable of transporting material across the transmembrane channel supporting the prediction that arcs play a dominant role in the leakage. Arc-like transmembrane constructs have been purported to exist for other a-helical transmembrane proteins such as actinoporins.…”

Assessing the Extent of Structural and Dynamic Modulation of Membrane Lipids due to Pore Forming Toxins: Insights from Molecular Dynamics Simulations

“…26 Disordered N-terminal residues 1-7 and the C-terminal residues 293-303 unresolved in the crystal structure are important for lytic activity [27][28][29] and hence were modeled using the I-TASSER web server 30,31 as described previously. 29,32 The heptameric crystal structure of the α-hemolysin (AHL) pore (PDB ID 7AHL) 33 has multiple residues with missing co-ordinates (arginine 66 and lysine 70 in chain A, lysine 30 and lysine 240 in chain D, lysine 283 in chain F and lysine 30 in chain G). The missing atoms in these residue side chains were reconstructed using VMD 1.9.1 34 similar to a previous study.…”

Membrane Perforation by the Pore-Forming Toxin Pneumolysin

“…Molecular dynamics (MD) simulations offer a promising alternative to resolve pore formation at the molecular scale. Previous MD simulations of αand β-PFTs (20)(21)(22)(23)(24)(25) were limited to either protein monomers or small oligomers. Simple rate models for the assembly and insertion process have been proposed (26,27), but the role of lipids and the process of pore opening have not been studied in detail.…”