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

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential mag nesium complexed to nucleotide. We have characterized mutations at this pos ition to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabi lized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S), Bind ing Phe-tRNA(Phe) to poly(U) ribosome needed a higher magnesium concentrati on 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-tRNA(Phe) against spontaneous hyd rolysis 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 riboso mes and kirromycin suggest that stimulation by these two agents follows dif ferent mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable com plex with EF-Ts than wild type. This implies that stabilization of the EF-T u 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-2 5-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.