Team:IISER Bhopal/Model Module2.html

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Smac-R8 Internalisation: MD simulations

After the fusion protein gets cleaved by metalloproteases into Smac(n7)-R8 and fusion TRAIL, the Smac(n7)-R8 fusion peptide must internalise inside the cancer cell’s cytoplasm. The fusion peptide contains a CPP (cell-penetrating peptide), polyarginine having eight residues in our case, to assist Smac(n7)’s entry into the cell. Due to delayed wet lab access, we did molecular dynamics simulations to study the interaction of Smac(n7)-R8 and Smac(n7) and the plasma membrane of a cancer cell.

We first studied the composition of the lipid bilayer. We found that the cancer cells which are near the metastasis stage have asymmetric bilayer composition. Since our project is for the early stages of cancer, we went with an asymmetric lipid bilayer. We used the lipid composition given below.

Lipid Molecule Upper leaflet (%) Lower leaflet (%)
POPC 27 12.5
POPE 8.5 42
POPS 18 25
PSM 29.5 3.5
CHOL 17 17

We first tried to predict the structure of Smac(n7)-R8 and Smac(n7) using a python library modeller. We ran a 1μs MD simulation to see how the protein folds in the presence of water molecules and ions. We found that the protein takes the shape of a simple coil.


Our next step was to set up a lipid bilayer and protein system for the simulation. We used Martini Bilayer Maker to set up a coarse-grain simulation since we couldn’t afford all-atom simulations due to the delayed access to resources. We used GROMACS and CHARMM force fields for energy minimization, equilibration and production run. Prior to full simulation, we are using steered MD simulations (SMD) to obtain windows in the reaction coordinates that can be used for umbrella sampling. This step is still in progress.

Initially, we set up the system using 120 lipids on each bilayer leaflet consisting of the lipids mentioned above in appropriate ratios. We introduced polar water molecules and ions on either side of the lipid bilayer.

The figure shows the setup for the coarse-grain simulations. We placed the protein around 45-50 Å from the centre of the lipid bilayer and gave different orientations to keep the results unbiased. Our first step was to check the interaction of the fusion peptide with the lipid bilayer. We ran simulations for both Smac(n7)-R8 and only Smac(n7).

Image: Coarse-grain simulation setup

Colour code for the above image:

  • Purple - Water molecules
  • Dark blue and Pink - Head of lipids
  • Aqua green - Tail of lipids

    • Due to time constraints, we could only simulate the initial interaction of Smac(n7)-R8 and Smac(n7) with the lipid bilayer.

    Due to delayed access to the computational resources, umbrella sampling of protein-lipid bilayer translocation simulations is still in progress.

    References

  • T. Rivel, C. Ramseyer, S. O. Yesylevskyy, Permeation of cisplatin through the membranes of normal and cancer cells: a molecular dynamics study.
  • Shahane, G., Ding, W., Palaiokostas, M. et al. Physical properties of model biological lipid bilayers: insights from all-atom molecular dynamics simulations. J Mol Model 25, 76 (2019). https://doi.org/10.1007/s00894-019-3964-0
  • Marco Klähn, Martin Zacharias, Transformations in plasma membranes of cancerous cells and resulting consequences for cation insertion studied with molecular dynamics.
  • Delin Sun, Jan Forsman, and Clifford E. Woodward, Atomistic Molecular Simulations Suggest a Kinetic Model for Membrane Translocation by Arginine-Rich Peptides.
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