Conformational changes in human prolyl-tRNA synthetase upon binding of the substrates proline and ATP and the inhibitor halofuginone

Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its effects by acting as a high-affinity inhibitor of the enzyme glutamyl-prolyl-tRNA synthetase (EPRS). In order to elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyl-tRNA synthetase domain of human EPRS ( h PRS) at 2.4 Å resolution ( h PRS-apo), of h PRS complexed with ATP and the substrate proline at 2.3 Å resolution ( h PRS-sub) and of h PRS complexed with HF at 2.62 Å resolution ( h PRS-HF) are presented. These structures show plainly that motif 1 functions as a cap in h PRS, which is loosely opened in h PRS-apo, tightly closed in h PRS-sub and incorrectly closed in h PRS-HF. In addition, the structural analyses are consistent with more effective binding of h PRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development of more potent and selective h PRS inhibitors for promoting inflammatory resolution.