Classical molecular dynamics

The tertiary structure of the linearized TC3 peptide was constructed in silico by truncating the predicted DefMT3 Ixodes ricinus salivary defensin from our previous study⁹. Both termini of the TC3 were capped with an acetyl group (amine terminus) and a N-methyl amide group (carboxyl terminus). The electrostatic potential of TC3 was generated using tools from the Schrodinger’s Maestro molecular software package, release 2020–4¹⁴. The CHARMM-GUI automated server¹⁵ was used to construct the heterogenous bilayer membrane. The upper leaflet of the membrane bilayer consisted of 62 phospholipids [32 phosphatidylcholine (POPC), 14 phosphatidylethanolamine (POPE), 6 phosphatidylinositol (POPI), 4 phosphatidylserine (POPS), 2 phosphatidic acid (POPA), 2 phosphatidylglycerol (POPG), and tetraoleoyl cardiolipin 2 (TOCL)]. The lower leaflet consisted of 64 phospholipids (36 POPC and 28 POPE). The heterogenous bilayer membrane therefore consisted of 126 phospholipids. The TC3 and membrane systems were then prepared separately and the hydrogen-bond network was optimized using the Schrodinger’s Maestro Protein Preparation Wizard¹⁶. After preparation and optimization, a global minimization was performed using Maestro default settings to remove any steric clashes.

The optimized TC3 peptide and bilayer membrane were oriented separately in an orthorhombic box (40 Å × 40 Å × 40 Å and 64 Å × 64 Å × 130 Å, respectively) with a TIP3P explicit model, neutralized and salted with 0.15 M NaCl. The force fields used to parameterize the two systems were, TIP3P CHARMM¹⁷ for the solvent, AMBER99SB-ILDN^18–20 for the TC3 peptide, and CHARMM36²¹ for the membrane and ions. The program CGenFF^22,23, based on CHARMM36²¹, was used to retrieve force field parameters for phospholipids POPI and TOCL. Classical molecular dynamics (MD) calculations were performed using Desmond²⁴. Semi-isotropic conditions for MD were used under an NPT ensemble coupled with a Nose–Hoover thermostat²⁵ and Martyna–Tobias–Klein barostat²⁶. The temperature was set at 37 °C with a RESPA²⁷ integrator at an inner time step of 2.5 fs. All MD calculations were conducted using a GPU-accelerated (i.e., graphics card) workstation.

The phospholipid bilayer membrane was first equilibrated using the Desmond²⁴ protocol provided by the CHARMM-GUI automated server¹⁵. The final CHARMM-GUI membrane production frame generated and the TC3 were separately equilibrated for 4 μs and 1 μs, respectively, using the MD protocol above. The final frame from the TC3 and membrane equilibration was used as the starting conformations for the combined peptide-membrane system. The TC3 was positioned 30 Å from its residue α-carbons to the phospholipid phosphorus atoms of the upper membrane surface and was equilibrated, as before, for 100 ns in an orthorhombic box 64 Å × 64 Å × 130 Å. Production MD calculations for analyses were then conducted for 1 μs using the last equilibrated frame under the same above protocol except using an NVT ensemble (where the volume is kept constant, not the pressure). The MD calculations for the cysteine oxidized form of TC3 (TC3Ox) was conducted using the same aforementioned protocol. The MD calculations were analysed using the Schrodinger’s Maestro software package¹⁴.