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Cartographie structurale et fonctionnelle de la liaison entre la peptidyl-ARNt hydrolase et son substrat

Abstract : Peptidyl-tRNA hydrolase (PTH) catalyzes the hydrolysis of peptidyl-tRNA molecules that have prematurely dissociated from the ribosome during protein translation. This protein is essential for bacterial viability whereas its eukaryotic homolog is dispensable. Therefore, PTH is an attractive target for the development of new antibacterials, and it seems important to map the interaction between this protein and its substrate in order to facilitate the design of inhibitors. Because attempts to obtain crystals of PTH with substrate mimics remained unsuccessful, we chose to study such complexes in solution, by NMR spectroscopy. Spectra of 15N-13C uniformly labeled Escherichia coli PTH allowed us to assign the resonance frequencies of the protein backbone atoms and of many side-chain atoms. Then, we analyzed the interaction between PTH and a chemically synthesized small molecule, 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, imitating the 3'-part of a peptidyl-tRNA. This study points out the role of many residues of the active center. In particular, the resulting enzyme-bound substrate model indicates (i) that a phenylalanine residue (Phe66) stacks with the adenine ring of the 3'-terminal adenosine, (ii) that an asparagine residue (Asn114) holds the water molecule involved in the hydrolysis of the substrate and (iii) that another asparagine residue (Asn10) confers to the PTH the capacity to distinguish peptidyl-tRNAs from aminoacyl-tRNAs. We also characterized the interaction between PTH and mini-tRNAs derived from the acceptor end of tRNAHis. These results confirmed the major role of Arg133 and Lys105 residues in the recognition of the 5'-phosphate of tRNA. They also showed an interaction between the C-terminal helix of the protein and the TΨC arm of the tRNA, 30 Å away from the active site. The functional relevance of the latter contact could be established by site-directed mutagenesis. Altogether, our results allow us to propose a complete model of the interaction between PTH and a peptidyl-tRNA.
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Submitted on : Tuesday, March 1, 2011 - 9:16:51 AM
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Giorgi Laurent. Cartographie structurale et fonctionnelle de la liaison entre la peptidyl-ARNt hydrolase et son substrat. Microbiologie et Parasitologie. Ecole Polytechnique X, 2010. Français. ⟨pastel-00572217⟩

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