Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Synthesis

Background:

• Elongation factor 2 (EF-2) is crucial for protein synthesis.
• ADP-ribosylation of EF-2 by certain toxins inhibits its function.

Purpose of the Study:

• To investigate the effect of ADP-ribosylation on guanosine nucleotide binding to wheat germ EF-2.
• To elucidate the mechanism by which ADP-ribosylation inhibits EF-2 activity.

Main Methods:

• Measurement of [3H]GDP and [gamma-32P]GTP binding to native and ADP-ribosylated EF-2.
• Assessing the influence of GTP and ribosomes on nucleotide binding.
• Evaluating EF-2 complex formation with GuoPP(CH2)P and ribosomes.

Main Results:

• ADP-ribosylation did not affect GDP binding to EF-2.
• [3H]GDP binding to native EF-2 was reduced by GTP and ribosomes, but not for ADP-ribosylated EF-2.
• GTP binding to EF-2 was significantly reduced by ADP-ribosylation.
• ADP-ribosylation abolished ribosome stimulation of GTP binding and high-affinity complex formation.

Conclusions:

• ADP-ribosylation impairs the ability of EF-2 to exchange GDP for GTP.
• This inability to perform nucleotide exchange is the likely mechanism for toxin-induced inhibition of protein elongation.