The Antibiotic Thermorubin Inhibits Protein Synthesis by Binding to Inter-Subunit Bridge B2a of the Ribosome

Abstract

Thermorubin is a small molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location - the interface between the small and large subunits that is formed by intersubunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA and presumably this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Theraputic Index > 200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin’s mode of action but possess improved pharmacodynamic properties.

Elements of ribosomal drug resistance and specificity

“…( A , B, C ) Overview of the superimposed binding sites of viomycin (purple) [18], paromomycin (red) [57], and thermorubin (yellow) [26] on the Thermus thermophilus 70S ribosome viewed from three different perspectives. Shown in light yellow is the 30S subunit with h44 in light orange and in light blue is the 50S subunit with H69 in marine.…”

“…Any disruption of this eubacteria-specific base-pair confers resistance to tuberactinomycins [14]. The same base-pair can explain the specificity of thermorubin for eubacterial ribosomes [26]. The tetracyclic moiety of thermorubin stacks against the bases of G1491and C1409 (Fig.…”

Selection of heptapeptides that bind helix 69 of bacterial 23S ribosomal RNA

“…Indeed, recent reports indicated that H69 is a target site for aminoglycoside antibiotics 15 and thermorubin. 15c Most naturally derived compounds have already given rise to resistance or will lead to resistance before long.…”

“…Indeed, recent reports indicated that H69 is a target site for aminoglycoside antibiotics 15 and thermorubin. 15c Most naturally derived compounds have already given rise to resistance or will lead to resistance before long. 1 The goal for this work was to discover new compounds, starting with small peptides that specifically bind to H69.…”

Allosteric control of the ribosome by small-molecule antibiotics

“…Rearrangements at the subunit interface have been implicated in the joining of large and small subunits during initiation and the positioning of tRNA substrates during processive elongation reactions, as well as the binding of translation factors during termination and the release of the protein products 23, 24 . Conformational plasticity of the centrally located intersubunit bridge B2, formed by the interaction of H69 of the large subunit with h44 of the small subunit, has been posited to function as a potential regulator of translation processes 25, 26 . Recently reported ribosome structures have revealed that remodeling in this region—immediately proximal to the tRNA and mRNA binding sites—facilitates rotation of the small subunit with respect to the large subunit.…”