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Identification of borrelidin binding site on threonyl-tRNA synthetase

Li, Ming, Zhang, Ji, Liu, Chongxi, Fang, Baozhu, Wang, Xiangjing, Xiang, Wensheng
Biochemical and biophysical research communications 2014 v.451 pp. 485-490
Escherichia coli, active sites, adenosine triphosphate, binding sites, bioactive properties, enzyme kinetics, fluorescence, molecular models, mutants, site-directed mutagenesis, threonine, threonine-tRNA ligase
Borrelidin exhibits a wide spectrum of biological activities and has been considered as a non-competitive inhibitor of threonyl-tRNA synthetase (ThrRS). However, the detailed mechanisms of borrelidin against ThrRS, especially borrelidin binding site on ThrRS, are still unclear, which limits the development of novel borrelidin derivatives and rational design of structure-based ThrRS inhibitors. In this study, the binding site of borrelidin on Escherichia coli ThrRS was predicted by molecular docking. To validate our speculations, the ThrRS mutants of E. coli (P424K, E458Δ, and G459Δ) were constructed and their sensitivity to borrelidin was compared to that of the wild-type ThrRS by enzyme kinetics and stopped-flow fluorescence analysis. The docking results showed that borrelidin binds the pocket outside but adjacent to the active site of ThrRS, consisting of residue Y313, R363, R375, P424, E458, G459, and K465. Site-directed mutagenesis results showed that sensitivities of P424K, E458Δ, and G459Δ ThrRSs to borrelidin were reduced markedly. All the results showed that residue Y313, P424, E458, and G459 play vital roles in the binding of borrelidin to ThrRS. It indicated that borrelidin may induce the cleft closure, which blocks the release of Thr-AMP and PPi, to inhibit activity of ThrRS rather than inhibit the binding of ATP and threonine. This study provides new insight into inhibitory mechanisms of borrelidin against ThrRS.