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.

Figure 1. (Fo–Fc) difference electron density map calculated at 2.5 σ cut off before adding water oxygen atoms in the structure of unbound protein (A), cytidine in the structure of the complex with protein (B) and uridine in the structure of the complex with protein (C).

Figure 2. Binding curves (A) for the binding of cytidine to AbPth and (B) for the binding of uridine to AbPth showing the changes in fluorescence intensities (ΔF/Fo) at 347 nm as the ligands to protein ratios were increased (5 µl, 10 µl, 15 µl and 20 µl).

The errors on the experimental points are indicated.

Figure 3. The SPR sensograms for the bindings of (A) cytidine and (B) uridine to protein, AbPth.

The protein was immobilized on CM-5 chip and the increasing concentrations (a) 0.8 µM, (b) 1.5 µM, (c) 2.5 µM and (d) 4.5 µM of analytes cytidine and uridine were used in mobile phase in separate experiments corresponding to curves in (A) and (B) respectively.

Figure 4. Showing overall folding of the polypeptide chain of AbPth.

The secondary structure elements are indicated where α-helices, α1 (residues: 24–36), α2 (residues: 72–82), α3 (residues: 116–125), α4 (residues: 146–151), α5 (residues: 156–176) and α6 (residues: 180–188) are shown in red and β-strands, β1 (residues: 5–10), β2 (residues: 41–43), β3 (residues: 48–52), β4 (residues: 58–64), β5 (residues: 89–96), β6 (residues: 103–108) and β7 (residues: 130–136) are shown in green. The N-terminal and C-terminal residues are also indicated.

Figure 5. The hydrogen bonded network between protein atoms and water molecules as well as between water molecules in the active site region.

Figure 6. The superimpositions of the C^α traces of AbPth (green), EcPth (cyan), PaPth (pink), MtPth (yellow) and MsPth (grey).

The flexible regions consisting of residues, (Met1 - Leu6), (Pro65 - Ala80), (Gly111 - Leu118), (Asp120 - Pro127), (His138 - Val146) and (Pro181 - Ala193) are marked by dotted circles.

Figure 7. Showing structures of (A) native AbPth with a hydrogen bonded water molecule W1 with both His22 and Asn70.

(B) AbPth with bound cytidine and (C) AbPth with uridine. The dotted lines indicate hydrogen bonds.

Figure 8. The distances between Asp98 O^δ2 and Gly113 O and His22N^ε2 and Asn70 O^δ1 in the structure of the (A) unbound protein and in its complexes with (B) cytidine and (C) uridine.

Figure 9. Structural environment of the segment Pro 65 - Pro 78 where the conformation of Tyr68 is observed in (A) disallowed region in AbPth (b) disallowed region in EcPth while the corresponding residues were found with allowed conformations in (C) PaPth, (D) MtPth and (E) MsPth.

Figure 10. Grasp representation of the fitting of (A) cytidine and (B) uridine in the active site pocket of AbPth.