Piper

The energy-minimized structures were subjected to MD simulation in a periodic boundary condition using the Desmond [64 ] (Schrödinger’s maestro, Desmond, NY, USA) software package with the OPLS3e force field. The systems were solvated with multiple layers of water molecules using the TIP3P water model. Further, the system was ionized and neutralized with 0.15 M of NaCl. The temperature and pressure were maintained using a Noose–Hover thermostat and Martyna–Tobias–Klein barostat respectively using a timestep of 2 fs. The cut-off distance to coulombic interaction was set to 9.0 Å. The monomers were simulated at various temperatures ranging from 300 K to 450 K for 50 ns. A choice of the temperature and time threshold was created to produce a partially unfolded protein structure. To study protein aggregation, the evolved protein structure was docked using the PIPER (Schrödinger, maestro) [65 (link),66 (link),67 (link)], where ten thousand conformations were generated, and the output was ranked according to the size of the cluster. The docked structures with the largest cluster size were selected. Structures were energy minimized and followed by a simulation of 200 ns at room temperature (300 K).

Molecular models were generated using Modeller[59 (link)] and MacroModel (Schrodinger, New York, NY). PfUCH37 was modelled using an ensemble of available UCH37 X-ray crystal structures (PDB IDs: 3A7S and 3IHR, 44% and 36% sequence identity respectively), including the complex of TsUCH37 with ubiquitin (37% sequence identity; PDB ID: 4IG7). The structures of PfUCH37 and TsUCH37 (PDB ID: 4IG7) were minimized using the MMF94s forcefield in Sybyl-X 2.1.1 (Certara L.P., St Louis, MO), with the final structure having more than 95% of residues in the allowed region of a Ramachandran plot. Ubiquitin and Nedd8 were docked into the structures of UCH37 using Piper (Schrodinger, New York, NY), with the available X-ray crystal structure of TsUCH37 in complex with ubiquitin (PDB ID: 4IG7) used to guide protein docking. The models of the complexes were minimised using the MMF94s forcefield in Sybyl-X 2.1.1 as described above. The quality of all the models were confirmed with Verify3D[60 (link)]. The structural consequences of the differences in interfacial residues were analysed to assess the structural importance of the residues[41 (link),61 (link)–63 (link)]. Interactions were calculated using Arpeggio[64 (link)] and model structures were examined using Pymol.