The importance of P-loop and domain movements in EF-Tu for guanine nucleotide exchange

Abstract

Elongation factor Ts (EF-Ts) is the guanine nucleotide exchange factor for elongation factor Tu (EF-Tu). An important feature of the nucleotide exchange is the structural rearrangement of EF-Tu in the EF-Tu.EF-Ts complex caused by insertion of Phe-81 of EF-Ts between His-84 and His-118 of EF-Tu. In this study, the contribution of His-118 to nucleotide release was studied by pre-steady state kinetic analysis of nucleotide exchange in EF-Tu mutants in which His-118 was replaced by Ala or Glu. Intrinsic as well as EF-Ts-catalyzed release of GDP/ GTP was affected by the mutations, resulting in an similar to 10-fold faster spontaneous nucleotide release and a 10-50-fold slower EF-Ts-catalyzed nucleotide release. The effects are attributed to the interference of the mutations with the EF-Ts-induced movements of the P-loop of EF-Tu and changes at the domain 1/3 interface, leading to the release of the beta-phosphate group of GTP/GDP. The K-d for GTP is increased by more than 40 times when His-118 is replaced with Glu, which may explain the inhibition by His-118 mutations of aminoacyl-tRNA binding to EF-Tu. The mutations had no effect on EF-Tu-dependent delivery of aminoacyl-tRNA to the ribosome.