Geometric placement of fentanyl in scaffolds using patchdock and matching

Scaffold proteins were used for initial docking of the set of fentanyl conformers. Docking was restricted to binding pockets identified by preexisting ligands in the crystal structure, or by RosettaHoles (Sheffler and Baker, 2009), which was used to define a position file of residues in the pocket. The 2.0 drug module of PatchDock (Schneidman-Duhovny et al., 2005) was used with the default settings for docking. The top 20 scoring poses for each scaffold were selected for subsequent RosettaDesign.~/Rosetta/main/source/bin/gen_apo_grids.linuxgccrelease -s PDBFILE -database ~/Rosetta/main/database @flags

Where the flags file contained the following (text following # are comments):-mute all -unmute apps.pilot.wendao.gen_apo_grids -chname off -constant_seed -ignore_unrecognized_res -packstat:surface_accessibility -packstat:cavity_burial_probe_radius 3.0 # if the cavity ball can be touched by probe r>3, then it is not in a pocket -packstat:cluster_min_volume 90 # minimum size of a pocket, smaller voids will not be considered -packstat:min_cluster_overlap 1.0 # cavity balls must overlap by this much to be clustered -packstat:min_cav_ball_radius 1.0 # radius of the smallest void-ball to consider -packstat:min_surface_accessibility 1.4 # voids-balls must be at least this exposed

These positions were set by using the receptorActiveSite in the PatchDock parameter file by initially converting the position file into PatchDock format:python splitfile.py PDBPOSITIONFILE.pos

Next, the parameters were generated using the supplied scripts: buildParams.plperl buildParams.pl PDBFILE FENTANYL_CONFORMATION 2.0 drug

The receptor active site was added to the parameter file:echo "receptorActiveSite POSITIONFILEPATCHDOCK" >> params.txt

Lastly, fentanyl was docked into each scaffold protein:patch_dock.Linux params.txt patchdock.out

For a small subset of the final 62 fentanyl binder designs, the RosettaMatch algorithm (Zanghellini et al., 2006) was used to introduce specific protein–fentanyl interactions to the subset of ketosteroid isomerase scaffold proteins (83 PDBs). Polar residues were used to introduce hydrogen bonds to the fentanyl carbonyl, while acidic (Asp and Glu) and aromatic residues (Phe, Trp and Tyr) were used to make charge–charge or dipole–quadrupole interactions, respectively, with the tertiary amine. A summary of the designs, their sequences and the method used to generate them is given in Supplementary Table 2 (Supplementary file 2).