Functional-structural analysis of threonine 25, a residue coordinating the nucleotide-bound magnesium in elongation factor Tu

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.