Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Many pathogenic bacteria secrete mono-ADP-ribosylating toxins.
• These toxins modify intracellular proteins, including translation elongation factor 2 (eEF2).
• Understanding the mechanism of ADP ribosylation is key to developing therapeutics.

Purpose of the Study:

• To elucidate the structural basis of ADP ribosylation by Pseudomonas aeruginosa exotoxin A (ETA).
• To investigate the role of the diphthamide residue in eEF2 during the enzymatic reaction.
• To understand the universal recognition mechanism of eEF2 by ETA.

Main Methods:

• Determined four crystal structures of a catalytically active complex between ETA and eEF2.
• Utilized a non-hydrolysable NAD+ analogue (betaTAD) to trap the reaction intermediate.
• Analyzed the structural interactions between ETA, eEF2, and betaTAD.

Main Results:

• The diphthamide residue in eEF2 spans a cleft and interacts with the NAD+ analogue.
• Diphthamide appears critical for triggering NAD+ cleavage and stabilizing the reaction intermediate.
• The bound NAD+ analogue mimics rRNA nucleotides, explaining ETA's broad recognition of eEF2.

Conclusions:

• Diphthamide is essential for ADP ribosylation by ETA, likely by facilitating NAD+ cleavage.
• ETA recognizes eEF2 through a mechanism involving structural mimicry of rRNA.
• The findings may inform understanding of similar toxins, such as diphtheria toxin.