Abstract
Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.
Highlights
Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu)
An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics
Aurodox (Fig. 1) shows identical effects to the more thoroughly studied kirromycin (3). These antibiotics have been shown to interfere with the role of bacterial elongation factor Tu (EF-Tu)1 in protein biosynthesis (4)
Summary
Preparation of the Ternary Complex EF-Tu1⁄7GDP1⁄7Aurodox—Since aurodox was reported to be moderately sensitive to light (21), exposure was kept at a minimum. Thereafter, refinement was continued with the model derived from the T. thermophilus EF-Tu1⁄7GppNHp complex only At this stage of refinement, the linker between the C terminus of domain 1 and the N terminus of domain 2/3 could be modeled into the electron density. In comparison with the structure of EF-Tu1⁄7GppNHp (5), the conformations of several side chains had to be altered, most notably that of Arg-124 (residue numbering is according to T. thermophilus EF-Tu) After this first round of refinement, the Rfree dropped to 0.335, and the densities for the GDP and aurodox molecules were very clear. The program Hyperchem (Hypercube Inc., Waterloo, Canada) was employed to build an initial atomic model for aurodox, which was fitted into an Fo Ϫ Fc difference density map (contoured at 2.5 above the mean), using the program O (30) This step improved the Rfree to 0.305. The rotation angle was calculated from the superposition operator of the analyzed elements
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