Abstract
Peptidyl-tRNA hydrolase (Pth) hydrolyzes the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of aborted translation, to prevent cell death by recycling tRNA. Numerous studies have attempted to elucidate the substrate recognition mechanism of Pth. However, the binding mode of the peptidyl-A76 (3'-terminal adenosine of tRNA) moiety of the substrate to Pth, especially the A76 moiety, remains unclear. Here, we present the crystal structure of Thermus thermophilus Pth (TtPth) in complex with adenosine 5'-monophosphate (AMP), a mimic of A76. In addition, we show the crystal structure of TtPth in which the active site cleft interacts with the C-terminal three amino acid residues of a crystallographically related neighboring TtPth molecule. Superimposition of these two crystal structures reveals that the C-terminal carboxyl group of the neighboring TtPth molecule and the 3'-hydroxyl group of AMP are located in positions favorable for ester bond formation, and we present a TtPth⋅peptidyl-A76 complex model. The complex model agrees with many previous NMR and kinetic studies, and our site-directed mutagenesis studies support its validity. Based on these facts, we conclude that the complex model properly represents the interaction between Pth and the substrate in the reaction. Furthermore, structural comparisons suggest that the substrate recognition mode is conserved among bacterial Pths. This study provides insights into the molecular mechanism of the reaction and useful information to design new drugs targeting Pth.