Computational protocol: A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis

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Protocol publication

[…] Three simulations were prepared: native tetrameric wild-type TTR and both native and Lys48-cleaved S52P variant TTR. As residue 48 is located far from the two different dimer interfaces, the four subunits were considered as independent and cleavage was performed on each protomer, in order to increase sampling. Available crystal structures only include a dimer (Mangione et al, ), and inclusion of symmetry mates within the crystal led to the creation of a tetrameric arrangement. Cleavage of all four TTR monomers in S52P tetramer was reproduced by inserting COO− and NH3+ termini between residues Lys48 and Thr49. Based on their neighbourhood, in all systems histidine amino acids were ε-protonated. All systems were solvated in a box of TIP3P water, and neutralized with the addition of 0.15 M Na+ and Cl− ions. Final systems had a size of approximately 60,000 atoms. Molecular dynamics simulations were performed using the Amber99SB (Hornak et al, ) force field on NAMD2.9 molecular dynamics engine (Phillips et al, ) with SHAKE algorithm constraining heavy-atom distances, and PME treating the electrostatic interactions in periodic boundary conditions. All the systems were first minimized with 2,000 conjugate gradient steps, and subsequently simulated using a 2 fs timestep. Systems were first equilibrated in the nPT ensemble for 0.5 ns, with a 10 kcal/mol constrain on protein alpha carbons. 300 K and 1 Atm were imposed by Langevin dynamics, using a damping constant of 1/ps, a piston period of 200 fs and a piston decay of 50 fs. Constraints were subsequently removed, and systems were simulated in the nVT ensemble at 300 K for 1 ns. Finally, production runs for wild-type, S52P and S52P cleaved systems were run in the nPT ensemble (300 K and 1 Atm) for 522, 523 and 679 ns, respectively. From every production simulation, one structure per nanosecond was finally extracted and its secondary structure content assigned using STRIDE as implemented in VMD’s timeline tool (Humphrey et al, ). The percentage of conserved secondary structure elements in each subunit was computed by comparing any frame to the first one. Movie and rendering of protein structures have been produced with VMD. […]

Pipeline specifications

Software tools NAMD, VMD
Application Protein structure analysis
Diseases Amyloidosis