The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

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.

High‐resolution crystal structure of peptidyl‐tRNA hydrolase from Thermus thermophilus

“…In the Ramachandran plot, 180 of the 182 residues are in the preferred regions, Glu38 and Ala44 are in the allowed region, and only Tyr55 is in the disallowed region. However, the torsion angles of the equivalent residues of Tyr55 are also in the disallowed regions in some other Pths . The conformation of Tyr55 therefore seems to be reasonable.…”

Crystal structure of peptidyl‐tRNA hydrolase from a Gram‐positive bacterium, Streptococcus pyogenes at 2.19 Å resolution shows the closed structure of the substrate‐binding cleft

“…In contrast, the corresponding r.m.s. shifts among C α traces of Pth enzymes belonging to Gram‐negative bacteria [24–29] were found to be in the range of 0.7–1.4 Å. It showed that the path of the polypeptide chain of a Pth enzyme from a Gram‐positive bacterium differed slightly from those of Gram‐negative bacteria.…”

“…SpPth shows sequence identities ranging from 30% to 32% ( Fig. 1) with Pth enzymes of other bacteria whose structures are known [24][25][26][27][28][29][30][31]. The three-dimensional structure of SpPth has been determined at 2.19 Å resolution.…”

Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase fromSalmonella typhimurium

“…During crystallization trials with lower concentrations of Pth1 (10 or 20 mg ml À1 ), limited crystal formation was observed even after several weeks. At the high concentration (120 mg ml Schmitt et al, 1997), P. aeruginosa Pth1 (PDB entry 4fyj; Hughes et al, 2012), F. tularensis Pth1 (PDB entry 3nea; Clarke et al, 2011), M. smegmatis Pth1 (PDB entry 3p2j, Kumar et al, 2012) and Acinetobacter baumannii Pth1 (PDB entry 3wh4, Kaushik et al, 2013). The regions with high variations in tertiary structure between the enzymes are indicated with arrows.…”