The importance of P-loop and domain movements in EF-Tu for guanine nucleotide exchange.
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.
Dahl
Louise D
LD
Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Århus C, Denmark.
Wieden
Hans-Joachim
HJ
Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany.
Rodnina
Marina V
MV
Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany.
Knudsen
Charlotte R
CR
Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Århus C, Denmark. Electronic address: crk@bioxray.dk.
D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry; D006150:Guanine Nucleotides / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000502:physiology; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000737:chemistry
D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D009711:Nucleotides; D010649:Phenylalanine; D006639:Histidine; D010444:Peptide Elongation Factor Tu