Small alterations in 3D stucture of a protein can answer for important functional differences – EF-Tu STORY

Martin Holub, Jaroslav Weiser

Institute of Microbiology, Czech Academy of Sciences, Vídeňská 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. Decoding of the information in mRNA via codon/anticodon interaction is mediated by ternary complex consisting of EF-Tu GTP.aminoacyl-tRNA. Repetitive participation of EF-Tu in elongation cycle requires its interaction with a number of ligands, among them the most important are guanine nucleotides GDP and GTP. 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. In some organisms it has been assigned for very special functions, although the primary sequence of the protein is highly conserved through out prokaryotes.

Besides the role in translation, EF-Tu is proposed to function in other compartments of the cell metabolism and that may be the reason, why this protein is a subject of a number of post-translation modifications. 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. During last few years this protein was found to function as an adhesion factor, for instance EF-Tu of M. pneumoniae binds fibronectin as part of a virulence mechanism. Another example is EF-Tu of Lactobacillus johnsonii, which mediates attachment of these bacteria to intestinal epithelial cells and mucins and stimulates proinflammatory reactions.

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 have 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 method mentioned above and performed on them comparative studies in order to understand better 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. We sequenced tuf genes coding EF-Tu in both strains and performed comparative 3D modelling of their structures and those of other Streptomyces strains with emphasis on their surface structures.