Substitution of Glu378 in EF-Tu disrupts binding to KKL-55

Trans -translation is a target for the development of new antibiotics. The potential antibiotic lead compound KKL-55 binds to EF-Tu and inhibits trans -translation. Previous structural and biochemical studies showed that glutamate 378 in EF-Tu directly contacts bound KKL-55, but mutation of residue 378 to alanine had no effect on the equilibrium dissociation constant for binding of EF-Tu and KKL-55. Here, we found that a variant of EF-Tu with tryptophan at position 378 increases the K _d for binding of EF-Tu and KKL-55 by >6-fold, indicating that a larger side chain at this position is disruptive. The E378W variant decreased the amount of translation in vitro and no trans -translation could be detected with this variant. These data provide further evidence that residue 378 of EF-Tu forms part of the KKL-55 binding pocket and are consistent with a lack of spontaneous mutants resistant to KKL-55.