Structural Basis of the Action of Pulvomycin and GE2270 A on Elongation Factor Tu,

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Abstract
Pulvomycin inhibits protein synthesis by preventing the formation of the ternary complex between elongation factor Tu (EF-Tu)·GTP and aa-tRNA. In this work, the crystal structure of Thermus thermophilus EF-Tu·pulvomycin in complex with the GTP analogue guanylyl imino diphosphate (GDPNP) at 1.4 Å resolution reveals an antibiotic binding site extending from the domain 1−3 interface to domain 2, overlapping the domain 1−2−3 junction. Pulvomycin binding interferes with the binding of the 3‘-aminoacyl group, the acceptor stem, and 5‘ end of tRNA. Only part of pulvomycin overlaps the binding site of GE2270 A, a domain 2-bound antibiotic of a structure unrelated to pulvomycin, which also hinders aa-tRNA binding. The structure of the T. thermophilus EF-Tu·GDPNP·GE2270 A complex at 1.6 Å resolution shows that GE2270 A interferes with the binding of the 3‘-aminoacyl group and part of the acceptor stem of aa-tRNA but not with the 5‘ end. Both compounds, pulvomycin more markedly, hinder the correct positioning of domain 1 over domains 2 and 3 that characterizes the active form of EF-Tu, while they affect the domain 1 switch regions that control the EF-Tu·GDP/GTP transitions in different ways. This work reveals how two antibiotics with different structures and binding modes can employ a similar mechanism of action.