biochemical and mutational analysis of the eukaryotic translation elongation guanine nucleotide exchange factor eEF1Bα

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A107-A108
• Main Authors: Pittman, Yvette Renee, Valente, Louis, Jeppesen, Mads Gravers, Andersen, Gregers Rom, Patel, Smita, Kinzy, Terri Goss
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A107-d

Translation elongation requires the GTP‐dependent activity of the eEF1 complex, which in S. cerevisiae has three subunits: eEF1A, eEF1Bα, and eEF1Bγ. To sustain efficient protein synthesis, eEF1Bα functions as the guanine nucleotide exchange factor (GEF) of eEF1A to maintain the active GTP‐bound form. Stopped‐flow kinetic analysis demonstrates that eEF1Bα stimulates nucleotide exchange 700‐fold. Structures predict K205 of eEF1Bα promotes GEF activity by disruption of the Mg2+ binding site and the lethality of a K205A mutant is not due to a structural defect. The K205A mutant drastically reduces exchange activity, but the K205R mutation is functional in vivo with nearly wild type GDP dissociation rates. Mg2+ does not affect the exchange activity of K205R mutant, however K205A does not respond to the inhibitory effect of Mg2+ on exchange activity. This data demonstrates the role of K205 and Mg2+ in the nucleotide exchange on eEF1A. F163 of eEF1Bα binds to a hydrophobic pocket of eEF1A proposed to overlap the aa‐tRNA binding site. We hypothesized that the hydrogen bond between W130 and E192 of eEF1Bα allows proper conformation for binding stability to eEF1A and exchange activity, however, loss of this bond constraint allows compensation for the F163A defect. In vivo suppression analysis confirms this model. These mutations allow us to dissect the contributions of eEF1A:eEF1Bα binding on nucleotide exchange and aa‐tRNA binding, thereby providing us with specific insight on G‐protein regulation.