Computational protocol: Immunologic Function and Molecular Insight of Recombinant Interleukin-18

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Protocol publication

[…] A structure of human mature IL-18 (157 amino acids) was obtained from the RCSB protein data bank (www.rcsb.org), PDB identification code 1J0S () []. As 1J0S.pdb is an NMR structure, the 3rd conformer was chosen on the basis of it being the lowest RMSD among 20 conformers. All hydrogen atoms in the structure were then removed and the protonation state of amino acid at pH 7 was determined using PROPKA webtools []. The missing hydrogen atoms, with corrected protonation state, were then re-inserted using the Leap module in AMBER12 package [,]. Six IL-18 mutants (E6K, M33Q, M60Q, T63A, E6K+T63A and M33Q+M60Q) were prepared using the Visual Molecular Dynamics (VMD) package [] and the Leap module as auxiliary tools. All IL-18 protein was finally energy-minimized using the steepest descent method, under AMBER10 nonpolarizable force field parameters, for 2000 steps.The minimized protein was neutralized by either sodium (Na+) or chloride (Cl−) ion and solvated by TIP3P water molecules along with NaCl, yielding a concentration of 0.15 mol dm-3. This protein-solution system was equilibrated in an isothermal ensemble (NVT), using Langevin Dynamics as a thermostat set at 310 K (37°C). The harmonic potential was applied to all atomic positions of IL-18 with force constants of 200, 100, 50, and 20 kcal mol−1Å2, and a time step of 1 femtosecond (fs). The system was finally switched to an isobaric/isothermal (NPT) ensemble with a time step of 2 fs. A temperature of 310 K and pressure of 1.013 bar (1 atm) were regulated using a weak coupling algorithm [], in order to mimic an in vivo environment. The NPT simulation was carried out for 70 nanoseconds (ns) as a product run by a Particle mesh Ewald molecular dynamics simulator (PMEMD) to handle electrostatics calculations with a 12-Å cutoff, implemented in an AMBER12 package. The first 50 ns period was omitted as an equilibration phase, and 2000 equidistant snapshots from the last 20 ns simulation were taken for a configurational average and analysis. Simulations of wild-type and 6 mutated ILs followed the identical protocol. All IL-18 resulting structures were analyzed, compared, and visualized using ptraj module and VMD package. […]

Pipeline specifications

Software tools PROPKA, AMBER, VMD
Application Protein structure analysis
Organisms Komagataella pastoris, Homo sapiens
Diseases Neoplasms