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J. Biol. Chem., Vol. 281, Issue 28, 19457-19468, July 14, 2006

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Mg²⁺ and a Key Lysine Modulate Exchange Activity of Eukaryotic Translation Elongation Factor 1B^*

Yvette R. Pittman, Louis Valente, Mads Gravers Jeppesen, Gregers Rom Andersen¹, Smita Patel^¶2, and Terri Goss Kinzy^||13

From the Department of Molecular Genetics, Microbiology & Immunology, and the ^¶Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, the Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark, and ^||The Cancer Institute of New Jersey, New Brunswick, New Jersey 08903-2681

To sustain efficient translation, eukaryotic elongation factor B (eEF1B) functions as the guanine nucleotide exchange factor for eEF1A. Stopped-flow kinetics using 2'-(or 3')-O-N-methylanthraniloyl (mant)-GDP showed spontaneous release of nucleotide from eEF1A is extremely slow and accelerated 700-fold by eEF1B. The eEF1B-stimulated reaction was inhibited by Mg²⁺ with a K of 3.8 mM. Previous structural studies predicted the Lys-205 residue of eEF1B plays an important role in promoting nucleotide exchange by disrupting the Mg²⁺ binding site. Co-crystal structures of the lethal K205A mutant in the catalytic C terminus of eEF1B with eEF1A and eEF1A·GDP established that the lethality was not due to a structural defect. Instead, the K205A mutant drastically reduced the nucleotide exchange activity even at very low concentrations of Mg²⁺.A K205R eEF1B mutant on the other hand was functional in vivo and showed nearly wild-type nucleotide dissociation rates but almost no sensitivity to Mg²⁺. These results indicate the significant role of Mg²⁺ in the nucleotide exchange reaction by eEF1B and establish the catalytic function of Lys-205 in displacing Mg²⁺ from its binding site.

Received for publication, February 3, 2006 , and in revised form, April 25, 2006.

The atomic coordinates and structure factors (codes 2B7B and 2B7C) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Institutes of Health (NIH) Grant GM62789.

² Supported by NIH Grant GM51966.

³ To whom correspondence should be addressed: University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5450; Fax: 732-235-5223; E-mail: kinzytg{at}umdnj.edu.

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