Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Abstract

The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD ؉ but dependent upon pH. The lack of requirement for NAD ؉ to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model. The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity. The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues. The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states. Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2. These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.

Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae

“…The tip of domain IV with the diphthamide and the nucleotide-binding site in domain I are separated by 60 -70 Å depending on the conformation of eEF2. Furthermore, ADP-ribosylation does not induce any significant change in domain IV, which might be transmitted to domain I through domains III or V. This agrees with previous data that suggested that the binding of guanyl nucleotides does not affect the binding of eEF2 to ETA (37).…”

“…The catalytic fragment of ETA with a C-terminal His 6 tag (PE24H) was overexpressed and purified as described (37).…”

“…This assay measures the fluorescence resonance energy transfer between the AEDANS moiety in PE24H (donor) and the fluorescein on eEF2 or ADPR-eEF2 (acceptor) (Fig. 1A) (37). From the binding isotherms (Fig.…”

BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of Pseudomonas aeruginosa

“…Additional differences in the extracellular proteome were noted with respect to exotoxin A. Production of exotoxin A inhibits eukaryotic host cell protein synthesis by interacting with elongation factor-2, resulting in cell death [65]. While exotoxin A was detected in the supernatants of P. aeruginosa PAO1, its presence was limited to biofilms (Fig.…”

Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation

“…Furthermore, the crystal structure of the binary complex (enzyme and tetrasaccharide substrate) of A1‐III has been characterized . By examining the high‐resolution crystal structure of the binary complex, 3D model of AlgL and our experimental data, we can propose a reaction mechanism for AlgL . At present, the “Asn‐His‐Tyr catalytic triad” is considered to be important in polysaccharide lyases catalysis.…”