The Importance of P-loop and Domain Movements in EF-Tu for Guanine Nucleotide Exchange

Louise D Dahl,Hans-Joachim Wieden,Marina V Rodnina,Charlotte R Knudsen

doi:10.1074/jbc.m602068200

Louise D Dahl, Hans-Joachim Wieden + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m602068200

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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 approximately 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.

Highlights

Reaction is initiated by the binding of Elongation factor Ts (EF-Ts) to EF-Tu1⁄7GDP to form the EF-Tu1⁄7GDP1⁄7EF-Ts complex, which is unstable and rapidly dissociates into GDP and EF-Tu1⁄7EF-Ts
In vivo, the reaction is driven toward formation of active elongation factor Tu (EF-Tu) for two reasons: (i) the formation of GTP-bound EF-Tu is favored by the higher intracellular concentration of GTP over GDP (0.9 versus 0.1 mM, respectively [4]) and (ii) the high concentration of aa-tRNA shifts the equilibrium toward the GTP-bound state due to the formation of EF-Tu1⁄7GTP1⁄7aa-tRNA, with no effect on the kinetics of nucleotide exchange [2]
The interaction of EF-Tu1⁄7GDP or EF-Tu1⁄7GTP with EF-Ts can be described by two consecutive equilibria that represent the formation of the ternary complex and nucleotide release, respectively

Summary

Introduction

Reaction is initiated by the binding of EF-Ts to EF-Tu1⁄7GDP to form the EF-Tu1⁄7GDP1⁄7EF-Ts complex, which is unstable and rapidly dissociates into GDP and EF-Tu1⁄7EF-Ts. For ternary complex formation, 3.7 ␮M wild-type EF-Tu, 3.7 ␮M EF-TuH118A, or 7.4 ␮M EF-TuH118E mutant was incubated with 1.9 ␮M tRNAPhe(proflavin 16/17), 3 mM ATP, 1 mM phenylalanine, 0.5% v/v yeast phenylalanyl-tRNA synthetase, 0.05 ␮M EF-Ts, 3 mM phosphoenolpyruvate, 1 mM GTP, 0.1 mg/ml pyruvate kinase in 50 mM Tris-HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 1 mM DTT at 37 °C for 45 min.

Results

Conclusion