Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu.
Several elongation factor (EF) Tu mutants (T25A, H22Y/T25S, D80N, D138N) that have impaired nucleotide binding show decreased solubility on overexpression in the E. coli cell, an indication that they do not fold correctly. Moreover, EF-Tu[T25A] and EF-Tu[D80N] were shown to inhibit cell growth on expression, an effect attributed to their sequestration of EF-Ts [Krab, I. M., and Parmeggiani, A. (1999) J. Biol. Chem. 274, 11132--11138; Krab, I. M., and Parmeggiani, A. (1999) Biochemistry 38, 13035--13041]. We present here results showing that the co-overexpression of EF-Ts at a 1:1 ratio dramatically improves the solubility of mutant EF-Tu, although in the case of EF-Tu[D138N]--which cannot at all bind the nucleotides available in the cell--this is a slow process. Moreover, with co-overexpression of EF-Ts, the mentioned growth inhibition is relieved. We conclude that for the formation of a correct EF-Tu structure the nucleotide plays an important role as a "folding nucleus", and also that in its absence EF-Ts can act as a folding template or steric chaperone for the correct folding of EF-Tu.
Krab
I M
IM
Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
te Biesebeke
R
R
Bernardi
A
A
Parmeggiani
A
A
D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006131:Growth Inhibitors / Q000737:chemistry; D006150:Guanine Nucleotides / Q000737:chemistry*; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000096:biosynthesis; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012263:Ribonucleosides / Q000235:genetics; D012995:Solubility; D014970:Xanthines
D006131:Growth Inhibitors; D006150:Guanine Nucleotides; D018832:Molecular Chaperones; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D012263:Ribonucleosides; D014970:Xanthines; C023512:elongation factor Ts; C005893:xanthosine; D005982:Glutathione Transferase; D010444:Peptide Elongation Factor Tu