Computational protocol: Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system

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Protocol publication

[…] Glide software (Schrödinger, Inc.) was used for the small molecule docking in this study. We first validated the performance of Glide against flexible molecules such as ATP and TCA [IUPAC name (2S,3R)-2-[[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxycarbonylamino]-3-hydroxybutanoic acid)] that contain 10 and 12 freely rotatable bonds, respectively. To do so, we extracted the ligand (ATP) from the Kae1 crystal structure (PDB:2IVP) and docked it back into the apo-form of the protein. Before docking, geometries of the ligands were converted from 2D to 3D with LigPrep (Schrödinger, Inc.) using the OPLS2005 force field, and then the structures were ionized to pH 7.0 with Epik and energy minimized with default LigPrep settings. The receptor protein GRID was calculated for a large box that was defined in the presence of ATP at the binding site. A standard box size of 10 Å beyond each side of the ligand was used. Two H-bond constraints were defined that recognize the adenosine portion: the side-chain oxygen atom of Glu176 and the side-chain NH of Asn257, both of which are interactions observed in the 2IVP crystal structure. The hydroxyls of Thr12, Ser129 and Tyr280 located in the vicinity of the ligand were designated as rotatable groups. We used the default scaling of van der Waals radii of 0.8 for atoms with partial atomic changes <0.15. We generated 5000 poses during the docking run, and then selected the 400 best poses for postdocking minimization and saved the top five poses for each ligand. The best docking pose was close to that observed in the 2IVP structure (0.65 Å RMSD based on the all-atom superposition) and preserved all eight H-bonds between the protein and the ligand as well as coordination of the Fe2+ ion by the phosphate group of ATP.This positive validation result encouraged us to proceed further and dock TCA into the refined structure of Qri7 following the identical procedure. The side chain of residue R218 was converted to an alternative rotamer to prevent steric hindrance to TCA. The resulting structure of TCA in Qri7 overlays closely with the adenosine, sugar and the first phosphate moieties of ATP in the 2IVP structure with Kae1. […]

Pipeline specifications

Software tools Glide, LigPrep, Epik
Applications Drug design, Protein interaction analysis