(595b) Molecular Dynamics Study of the Cytolysin A Pore Forming Toxin Using Atomistic and Coarse Grained Simulations

Pore forming toxins are a unique class of proteins that assemble in biological membranes to form stable pores, which ultimately lead to cell lysis. Understanding the self-assembly and molecular mechanism of pore formation by these toxins, and their effect on the surrounding membrane has implications in disease control, cancer and gene therapies. Cytolysin A (ClyA) is a membrane-associated cytolytic protein expressed by Escherichia coli and other enterobacteria. It is secreted in a water soluble form, and the hypothesized mechanism is that it inserts into the membrane, undergoes a conformational change, and oligomerizes on the surface of the membrane. Using a recently reported surfactant crystallized structure of the dodecameric pore-complex formed by ClyA, we carried out all-atom (CHARMM 27), united-atom (GROMOS 54A7) and coarse-grained (MARTINI) molecular dynamics simulations of the assembled pore complex in a POPC membrane. These multi-scale molecular dynamics simulations enable us to address various aspects of the pore-complex and its effect on the lipid membrane surrounding it.

In the all atom simulations, the pore structure is found to be stable over a 140 ns simulation, independent of the salt content. The all-atom simulations indicate a two fold increase in the overall number of salt bridges in the membrane pore complex when compared with the crystal structure data, suggesting increased stabilization of the pore complex in the membrane. The MARTINI simulations were carried out for a time duration of 5 microseconds to further verify the stability of the pore complex in the membrane. The united-atom simulations were run for a total of 120 ns. Using these relatively long time scale simulations we analyzed the lipid dynamics around the pore complex and observed the effect of the pore-complex on the lipids in its vicinity. United-atom and MARTINI simulations of the intermediate oligomers shed light on the pore forming mechanism. In the absence of crystal structures of these intermediate oligomers, these models can be used to obtain valuable insights into the collective lipid movement and dynamics around these complexes. The coarse-grained simulations show rapid evacuation of the lipids from the interior of the octamer and higher level intermediates to the bulk membrane, thus suggesting that lipid evacuation occurs well before the final stage of the dodecamer oligomerization.