Protein-protein interactions and inhibition of the ADP-ribosyl transferase reaction of Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety from NAD+ to its target protein, eukaryotic elongation factor 2 (eEF2). The objective of this research was to improve the understanding of the interactions between the catalytic domain of ETA and both of its substrates, eEF2 and NAD +. Through a study of water-soluble compounds, the inhibition of the catalytic domain of ETA was evaluated. These compounds mimic nicotinamide and the more potent inhibitors contained planar ring systems. PJ34 was further characterized and shown to act as a competitive inhibitor and was co-crystallized with the toxin to 2.1 A resolution, whereby important hydrogen bonds and van der Waals interactions were identified. An HPLC-based NAD+ -glycohydrolase assay was developed and showed that the NAD +-analogue, 2'-F-ribo-NAD+, was a competitive inhibitor and not a competing substrate, as it was hydrolyzed at 0.2% of the rate for NAD+. Next, the binding between ETA and eEF2 was investigated. The binding of eEF2 to the toxin is pH-dependent and correlates with the pH profile for catalytic function. Also, eEF2 retained native binding affinity for the toxin when it was ADP-ribosylated or when it bound a guanyl nucleotide, illustrating that eEF2 does not undergo large structural changes that would disrupt the eEF2-toxin binding site. A fluorescence study identified the molecular contacts between the catalytic domain of ETA and eEF2. The change in wavelength emission maxima, lifetime and acrylamide quenching for the IAEDANS fluorophore conjugated to the toxin was determined before and after eEF2 bound. The contact between these proteins is minimal and occurs primarily near the active site and a loop that modulates the transferase activity of the toxin. A fluorescence resonance energy transfer (FRET) technique was used to measure the energy transfer efficiencies between the acceptor fluorophore, fluorescein, on eEF2 at position 574 and the various positions of the donor fluorophore, IAEDANS, on the toxin. Distances between the donor-acceptor pair were calculated from the efficiency values and used to develop a FRET model for this apo-toxin-eEF2 complex. Moreover, the addition of beta-TAD, an NAD+ analogue, results in closer association of these proteins as indicated by increased efficiency values.