After the homology models were built, the docking study was performed to explore the best possible modes of ligand binding with LMP2 (i.e. β1i) subunit of immunoproteasome and β5 subunit of constitutive proteasome using the Glide. Glide (Grid-Based Ligand Docking with Energetics) approximates a systematic search of positions, orientations, and conformations of the ligand in the receptor binding site using a series of hierarchical filters.34 (link),38 (link) The shape and properties of the receptor are represented on a grid by several different sets of fields that provide progressively more accurate scoring of the ligand pose.
To facilitate the definition of the center of the active site for docking, the crystal structure complex of regular proteasome with epoxomicin (PDB code: 1G65) was used, as neither the X-ray crystal structure of regular proteasome β5 subunit (PDB code: 1IRU-L) nor the homology modeled LMP2 subunit have a ligand bound in the active site. First, the two covalent bonds between Thr 1 and epoxomicin were broken and the missing atoms of epoxomicin were added. Then, the complex (1G65-KL) with waters removed was minimized in vacuum using Amber program (version 8) with all the residues fixed except the ligand in order to mimic the conformation of the ligand before reactions. After that, the minimized complex substructure 1G65-K was aligned to 1IRU-L and LMP2 by superimposing the backbone atoms in order to transfer the epoxomicin into 1IRU-L and LMP2 to get a reference ligand for docking. The RMSD values of the alignments for the 1IRU-L and LMP2 are 1.28 and 1.25 Å, respectively. After alignments, the coordinates of epoxomicin were transferred into 1IRU-LM and LMP2/MECL1. For the histidine residues, hydrogens were placed at the ε-position for His 35 in LMP2, His 66, His 97, and His 185 in MECL1; His 36 and His 58 in 1IRU-M; and at the δ-position for His 66, His 93, His 114, and His 116 in LMP2; His 38 in MECL1; His 10, His 178, and His 196 in 1IRU-L; His 77 and His 163 in 1IRU-M. Then, the two complexes (1IRU-LM and LMP2/MECL1 with epoxomicin in the active site) were minimized in vacuum with all the residues fixed except the ligand to get the best position of the ligand.
The proteins were imported into Maestro and prepared in the presence of ligand in the active site using Pprep in Glide. The grid midpoint was defined by the center of the bound epoxomicin. Geometries of both ligands were optimized at Hartree-Fock (HF) level with 6–31G* basis set using Gaussian program (03 version)37 and were docked with Glide in extra-precision mode with up to thirty poses saved per molecule. For each molecule, the best scoring pose was selected for the subsequent molecular dynamics simulations and binding free energy calculations.
To facilitate the definition of the center of the active site for docking, the crystal structure complex of regular proteasome with epoxomicin (PDB code: 1G65) was used, as neither the X-ray crystal structure of regular proteasome β5 subunit (PDB code: 1IRU-L) nor the homology modeled LMP2 subunit have a ligand bound in the active site. First, the two covalent bonds between Thr 1 and epoxomicin were broken and the missing atoms of epoxomicin were added. Then, the complex (1G65-KL) with waters removed was minimized in vacuum using Amber program (version 8) with all the residues fixed except the ligand in order to mimic the conformation of the ligand before reactions. After that, the minimized complex substructure 1G65-K was aligned to 1IRU-L and LMP2 by superimposing the backbone atoms in order to transfer the epoxomicin into 1IRU-L and LMP2 to get a reference ligand for docking. The RMSD values of the alignments for the 1IRU-L and LMP2 are 1.28 and 1.25 Å, respectively. After alignments, the coordinates of epoxomicin were transferred into 1IRU-LM and LMP2/MECL1. For the histidine residues, hydrogens were placed at the ε-position for His 35 in LMP2, His 66, His 97, and His 185 in MECL1; His 36 and His 58 in 1IRU-M; and at the δ-position for His 66, His 93, His 114, and His 116 in LMP2; His 38 in MECL1; His 10, His 178, and His 196 in 1IRU-L; His 77 and His 163 in 1IRU-M. Then, the two complexes (1IRU-LM and LMP2/MECL1 with epoxomicin in the active site) were minimized in vacuum with all the residues fixed except the ligand to get the best position of the ligand.
The proteins were imported into Maestro and prepared in the presence of ligand in the active site using Pprep in Glide. The grid midpoint was defined by the center of the bound epoxomicin. Geometries of both ligands were optimized at Hartree-Fock (HF) level with 6–31G* basis set using Gaussian program (03 version)37 and were docked with Glide in extra-precision mode with up to thirty poses saved per molecule. For each molecule, the best scoring pose was selected for the subsequent molecular dynamics simulations and binding free energy calculations.
