Probing the reactivity of the GTP- and GDP-bound conformations of elongation factor Tu in complex with the antibiotic GE2270 A.

The activity of Escherichia coli elongation factor Tu (EF-Tu) depends on its GTP- and GDP-bound conformations. In this work we have studied the influence of the antibiotic GE2270 A, a new EF-Tu-specific inhibitor of protein biosynthesis, on these two conformations with respect to the interaction with the various ligands and stability. One molecule of GE2270 A bound per EF-Tu is sufficient to block poly(U)-directed poly(Phe) incorporation. This drug binds stably to both EF-Tu.GTP and EF-Tu.GDP but only affects the reactivity of the GTP-bound conformation that is no longer able to interact efficiently with aminoacyl-tRNA (aa-tRNA) and ribosomes. Consequently, the protection by EF-Tu.GTP of the aminoacyl-ester bond against nonenzymatic hydrolysis is strongly weakened. The intrinsic EF-Tu GTPase is little affected, but its response to aa-tRNA and ribosomes is impaired. The specificity of GE2270 A to the GTP-bound form of EF-Tu is supported by the course of temperature- and urea-dependent inactivation or tryptic digestion. Furthermore, in its presence elongation factor Ts (EF-Ts) can enhance the dissociation of EF-Tu.GDP but not that of EF-Tu.GTP. Unlike kirromycin, this antibiotic can bind to EF-Tu.EF-Ts without causing dissociation of this complex. Evidence was obtained that the EF-Tu binding site for GE2270 A is also distinct from those for kirromycin, pulvomycin, and aa-tRNA, even though this antibiotic functionally interferes with all three of these ligands. GE2270 A appears to interact with a crucial regulatory region of the EF-Tu molecule that is activated in the GTP-bound state.