Molecular basis for drug- and peptide-dependent translational arrest
Structural Biology
Final Report Abstract
The translational machinery is a major target within the cell for antibiotics. Many clinically used classes of antibiotics, such as the tetracyclines (tigecycline) and macrolides (erythromycin, tylosin), inhibit translation by binding to the ribosome. Despite the potency of many of these drug classes, antibiotic resistance among clinically relevant pathogens is an increasing problem and thus the need for new antibiotics is more urgent than ever before. Many bacteria utilize methyltransferases to modify the ribosome to prevent drug binding and thereby confer resistance, such as Erm methyltransferases that modify A2058 to confer resistance to macrolide antibiotics. Expression of Erm methyltransferases is often regulated so that expression is only induced in the presence of the drug. The induction of expression requires translational stalling within an upstream open reading frames (uORFs), with stalling being dependent on the presence of the macrolide as well as the specific sequence of the uORF. Classic uORFs that stall in the presence of macrolides are ErmAL, ErmBL, ErmCL and ErmDL. In addition, antibiotic resistance can be conferred via ribosome protection proteins that bind to the ribosome and chase the drug from its binding site, such as TetM-mediated resistance to tetracycline antibiotics. The results of this proposal have shed light into both these types of resistance mechanisms. We have determined the structures of the four ErmAL- ErmDL classes and reveal that they have distinct mechanisms by which they evoke translational stalling in response to the presence of the macrolide. We have determined a structure of TetM in complex with the ribosome, revealing how TetM sterically dislodges tetracyclines from their binding site. Lastly, we have also determined structures of the orthosomycin antibiotics evernimicin and avilamycin on the ribosome, revealing that the binding site is located far from the binding site of all other clinically used antibiotics, and thus explaining their lack of cross-resistance. We believe that by understanding the structural details of the interaction of drugs with the ribosome and the mechanism by which bacteria obtain resistance, we will open new pathways for the development of improved antibiotics to overcome multi-drug resistant pathogenic bacteria.
Publications
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Drug sensing by the ribosome induces translational arrest via active site perturbation. Mol. Cell. 2014 Nov 6;56(3):446-52
Arenz S, Meydan S, Starosta AL, Berninghausen O, Beckmann R, Vázquez-Laslop N, Wilson DN
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Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide. Nat. Commun. 2014 Mar 24;5:3501
Arenz S, Ramu H, Gupta P, Berninghausen O, Beckmann R, Vázquez-Laslop N, Mankin AS, Wilson DN
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Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution. Proc. Natl. Acad. Sci. USA. 2015 Apr 28;112(17):5401-6
Arenz S, Nguyen F, Beckmann R, Wilson DN
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Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling. Nat. Commun. 2015 Apr 23;6:6941
Sohmen D, Chiba S, Shimokawa-Chiba N, Innis CA, Berninghausen O, Beckmann R, Ito K, Wilson DN
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A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat. Commun. 2016 Jul 6;7:12026
Arenz S, Bock LV, Graf M, Innis CA, Beckmann R, Grubmüller H, Vaiana AC, Wilson DN
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Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome. Proc. Natl. Acad. Sci. USA. 2016 Jul 5;113(27):7527-32
Arenz S, Juette MF, Graf M, Nguyen F, Huter P, Polikanov YS, Blanchard SC, Wilson DN
