Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base.
Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.
Wieden
Hans-Joachim
HJ
Institute of Physical Biochemistry, University of Witten/Herdecke, 58448 Witten, Germany.
Gromadski
Kirill
K
Rodnin
Dmytro
D
Rodnina
Marina V
MV
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D010444:Peptide Elongation Factor Tu