Is EF-Tu as a well-understood bacterial molecular switch still Puzzle for structural biologists?

Martin Holub,  Jaroslav Weiser

Institute of Microbiology, Czech Academy of Sciences, Vídeòská 1083, 142 20 Prague 4, Czech Republic


Protein synthesis elongation factor Tu (EF-Tu) represents one of the major components of translation system in prokaryotes. It participates on the correct positioning of the incoming aminoacyl-tRNA on the ribosome where polypeptide chain is synthesised. Besides this, EF-Tu is proposed to function in other parts of the cell metabolism as well. The protein is represented by three-domain structure and behaves like a typical G (guanine nucleotide binding) protein. Interaction of flexible domain 1 containing GDP/GTP binding pocket with more rigid domains 2 and 3 allows it to work as a molecular switch changing between “on” and “off” conformation upon binding of GDP or GTP. There are available specific inhibitors of EF-Tu, which are able to “freeze “the protein in either “on”, or “off” conformation, as an example can be mentioned kirromycin or pulvomycin. The protein is recognized as a classical cytoplasmic protein, however, thanks to some of its below listed features,  it may be considered   as a special case.

There have been described several post-translation modifications of the protein. Some of them are playing the role in translation, others are important for its potential functions outside of the elongation cycle. In E. coli, Bacillus subtilis and Bacillus licheniformis a part of EF-Tu population, which is located on the membrane, can be methylated in response to starvation for an essential nutrient.

EF-Tu from E. coli and T. thermophilus was found to be phosphorylated in vivo, and the phosphorylated fraction remained stable under different conditions. Since the phosphorylated residue (Thr-382) is conserved in all known EF-Tu corresponding sequences from other species, the phosphorylation might be a common phenomenon. EF-Tu was also described recently as a major component of cytoskeleton like structures in Mycoplasma pneumoniae cells and most importantly it was identified there as a protein anchored in the membrane and binding fibronectin which is a multifunctional protein interacting with molecular motor like structures in eukaryotes.

We described previously a spontaneous polymerisation of EF-Tu from Streptomyces aureofaciens, which might serve as a protective mechanism for EF-Tu present in spores or enables the protein to play a structural role. Aggregates are formed under physiological conditions and give raise to filamentous structures large enough to be visible in the light microscope. We have developed simple and effective method for purification of large amounts of the aggregated protein, which retains its nucleotide binding activity. We found that two closely related strains of Streptomyces aureofaciens contain EF-Tu capable of spontaneous aggregation in contrast to number of other Streptomyces species of which EF-Tu gene was cloned and protein isolated. We purified EF-Tu from both strains using above mentioned method and used them in comparative studies in order to better understand the structural and functional features of this phenomenon. Using 2D electrophoresis of purified proteins and their hydrolysis products we analysed their structural differences and heterogeneity resulting from their post-translation modifications.

For the future we will compare sequences of Tuf-1, gene coding active EF-Tu, from both strains mentioned above and we will try to find why EF-Tu from S. aureofaciens is capable of spontaneous aggregation.