Abstract
The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.
Highlights
Peptidyl-tRNA hydrolase (Pth) is involved in the processing of prematurely dissociated peptidyl-tRNAs from ribosome during protein biosynthesis into peptide and tRNA [1,2]
We report here the three-dimensional structures of unbound AbPth and two of its complexes with cytidine and uridine including binding studies using fluorescence spectroscopic and surface plasmon resonance studies
Three important goals for carrying out structural studies of peptidyl-tRNA hydrolase (Pth) were (i) to determine structural features of Pth including the stereochemical details of the active site, (ii) to explain the mechanism of catalytic action and (iii) to understand the mode of ligand binding for the design of inhibitors
Summary
Peptidyl-tRNA hydrolase (Pth) is involved in the processing of prematurely dissociated peptidyl-tRNAs from ribosome during protein biosynthesis into peptide and tRNA [1,2]. Pth acts upon the ester link in peptidyl-tRNA between the peptide and the 29 or 39 OH group at the 39 end of tRNA [3,4] The products of this reaction are reused in the cell in the ongoing process of protein biosynthesis in bacteria [5,6]. The design of tight inhibitors of AbPth will help in developing useful antibacterial drugs against infections caused by Acinetobacter baumannii. In this regard, it is fortunate that the structures of eukaryotic and prokaryotic peptidyl-tRNA hydrolases are not similar [6]. The design of potent inhibitors of prokaryotic peptidyl-tRNA hydrolases will be a good approach
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