Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa

Abstract

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant K(i) of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 A (1 A=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

Figure 1. Chemical structures of the ETA inhibitors

The inhibitors are grouped according to their core ring structures. The substrate NAD⁺ and the inhibitor NAP are shown for comparison.

Figure 2. Inhibitory properties of PJ34 against PE24H

(A) The Dixon plot for PJ34 in the presence of different fixed concentrations of substrate, ε-NAD⁺. The ADPRT activity of PE24H at 50 (☆), 100 (◇), 200 (△), 300 (○) and 500 (□) μM ε-NAD⁺. v₀, initial velocity. (B) The re-plot of the slopes of the Dixon plot where the slope is equal to K_M/(V_maxK_i). The K_M and V_max values for the NAD⁺-dependent ADPRT reaction are 121 μM and 26.7 pmol/s respectively. The assay included 20 mM Tris/HCl, pH 7.9, 0–500 μM ε-NAD⁺, 14 μM eEF2, 0–1200 nM PJ34 and 10 nM PE24H in a total volume of 70 μl. (C) Binding of PJ34 to PE24H by quenching of intrinsic protein fluorescence. The titration was conducted in 20 mM Tris/HCl, pH 7.9, 50 mM NaCl at 25 °C using 1.25 μM toxin. The fluorescence excitation was at 295 nm with fluorescence emission at 340 nm (at band passes set to 4 nm). The data were corrected for the dilution effect.

Figure 3. Structure of PE24H–PJ34

(A) Superposition of the modelled loop from the catalytic domain of ETA (PE24H) in complex with PJ34 [monomer A (shown in grey)] compared with the loops from previous ETA structures. The loop from the catalytic domain of ETA in complex with either β-TAD (shown in purple, monomer B; PDB number 1AER [8]) or hydrolysed NAD⁺ (shown in orange, monomer B; PDB number 1DMA [12] are illustrated. The main chain is shown from residues 456 to 465. (B) The PJ34 inhibitor bound within the active site of the toxin. The hydrophobic pocket is shown in yellow and the inhibitor in purple. This hydrophobic pocket comprises the residues His-440, Gly-441, Tyr-470, Ile-471, Ala-472, Leu-477, Ala-478 and Tyr-481. (C) The 2F_obs-F_calc omit map of PJ34 bound to monomer A within the active site of ETA contoured at 1σ. Phases for the map were calculated with no contribution from the PJ34 atoms. (D) The binding of the inhibitor PJ34 to PE24H. PJ34 is shown in green and important toxin residues involved in hydrogen bonding (broken lines) or hydrophobic interactions are shown.

Figure 4. Comparison of PE24H–PJ34 with the active sites from ETA (with β-TAD bound), DT and PARP

(A) Superposition of the structure of PE24H–PJ34 (shown in lime green) on the catalytic domain of ETA in complex with β-TAD, an NAD⁺-analogue (shown in black; PDB number 1AER [8]). β-TAD contains a thiazole substituent that mimics nicotinamide. (B, C) Superposition of the structure of PJ34–PE24H (shown in orange) with (B) DT (shown in pink; PDB number 1TOX [13]) or (C) PARP-NU1025 (shown in pink; PDB number 4PAX [21]). The segment that includes a portion of the catalytic loop of ETA (termed LOOP) is shown and encompasses residues 482–487 in ETA, 66–71 in DT or 908–913 in PARP. In the DT comparison (B), the PJ34 inhibitor (green) is shown, whereas in the PARP comparison (C) the PARP inhibitor NU1025, 8-hydroxy-2-methyl-3-hydro-quinazolin-4-one (green), is shown and the PJ34 ligand omitted, to demonstrate the same relative orientation of a PARP inhibitor within the toxin active site as PJ34.