Dimers of Thermus thermophilus elongation factor Ts are required for its function as a nucleotide exchange factor of...

Elongation factor Ts (EF-Ts) promotes the formation of active GTP-bound elongation factor Tu (EF-Tu) by accelerating the dissociation of GDP from the EF-Tu x GDP complex. Thermus thermophilus EF-Ts forms a dimer in solution, which is stabilised by interaction of a three-stranded antiparallel beta-sheet from each of the two EF-Ts molecules. A disulfide bridge and several hydrophobic interactions are the main structural elements which stabilise the dimer [Jiang, Y., Nock, S., Nesper, M., Sprinzl, M. & Sigler, P. B. (1996) Biochemistry 35, 10269-10278]. Site-directed mutagenesis was used to study the dimer formation and the effect of dimerization on the nucleotide exchange activity. The presence of the covalent disulfide bridge between the Cys190 residues has no effect on the activity. However, this disulfide bridge is not a necessary condition for the activity of EF-Ts. The amino acid residues Leu73, Cys190 and Phe192 form a hydrophobic core on the dimerization interface. Their replacement by Asp, Ala and Asp, respectively, influences to different degrees the stability of EF-Ts, the ability of EF-Ts to form dimers, and the ability to interact with EF-Tu. EF-Ts variants which were unable to form dimers were also inactive in the nucleotide exchange on EF-Tu. CD spectroscopy confirmed that this loss of activity is not due to changes in EF-Ts secondary structure. By calorimetric measurements, it was demonstrated that the dimer formation considerably contributes to the thermostability of T. thermophilus EF-Ts. Dimerization of T. thermophilus EF-Ts is required to fulfil its physiological function in protein biosynthesis and probably represents a strategy of the translation system in this thermophile to withstand high temperatures. The biochemical data presented here are supported by the recently solved structure of the T. thermophilus EF-Tu x EF-Ts complex [Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P. B. (1997) Nature Struct. Biol. 4, 650-656] in which each EF-Tu in the dyad symmetrical heterotetrameric complex interacts with two subunits of EF-Ts.