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A tale of two antibiotics: Fusidic acid and Viomycin
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits bacterial protein synthesis by binding to elongation factor G (EF-G) on the ribosome, thereby inhibiting translocation of the bacterial ribosome. Viomycin binds directly to the ribosome and inhibits both the fidelity of mRNA decoding and translocation. We found that the mechanisms of inhibition of these two antibiotics were unexpectedly complex. Fusidic acid can bind to EF-G on the ribosome during three separate stages of translocation. Binding of the drug to the first and most sensitive state does not lead to stalling of the ribosome. Rather the ribosome continues unhindered to a downstream state where it stalls for around 8 seconds. Dissociation of fusidic acid from this state allows the ribosome to continue translocating but it soon reaches yet another fusidic acid sensitive state where it can be stalled again, this time for 6 seconds. Viomycin inhibits translocation by binding to the pre-translocation ribosome in competition with EF-G. If viomycin binds before EF-G it stalls the ribosome for 44 seconds, much longer than a normal elongation cycle. Both viomycin and fusidic acid probably cause long queues of ribosomes to build up on the mRNA when they bind. Viomycin inhibits translational fidelity by binding to the ribosome during initial selection. We found that the concentration of viomycin required to bind to the ribosome with a given probability during decoding is proportional to the accuracy of the codon∙anticodon pair being decoded. This demonstrated that long standing models about ribosomal accuracy cannot be correct. Finally, we demonstrated that a common viomycin resistance mutation increases the drug binding rate and decreases its dissociation rate. Our results demonstrate that ribosome targeting drugs have unexpectedly complex mechanisms of action. Both fusidic acid and viomycin preferentially bind to conformations of the ribosome other than those that they stabilize. This suggests that determining the structures of stable drug-bound states may not give sufficient information for drug design.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1399
Keyword [en]
Protein Synthesis, Antibiotics, Fusidic acid, Viomycin, Translocation, Accuracy
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-300479ISBN: 978-91-554-9644-9OAI: oai:DiVA.org:uu-300479DiVA: diva2:951568
Public defence
2016-09-23, B41 BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-09-01 Created: 2016-08-09 Last updated: 2016-09-05
List of papers
1. Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
Open this publication in new window or tab >>Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, 3440-3454 p.Article in journal (Refereed) Published
Abstract [en]

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-247496 (URN)10.1074/jbc.M114.611608 (DOI)000349456000020 ()25451927 (PubMedID)
Available from: 2015-03-19 Created: 2015-03-19 Last updated: 2016-08-26Bibliographically approved
2. Molecular mechanism of viomycin inhibition of peptide elongation in bacteria
Open this publication in new window or tab >>Molecular mechanism of viomycin inhibition of peptide elongation in bacteria
2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, 978-983 p.Article in journal (Refereed) Published
Abstract [en]

Viomycin is a tuberactinomycin antibiotic essential for treating multi-drug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of similar to 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Keyword
protein synthesis, viomycin, ribosome, antibiotics, tuberculosis
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-277786 (URN)10.1073/pnas.1517541113 (DOI)000368617900047 ()26755601 (PubMedID)
Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2016-08-26Bibliographically approved
3. Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
Open this publication in new window or tab >>Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
(English)Article in journal (Refereed) Submitted
Abstract [en]

Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic viomycin on mRNA decoding. We find that viomycin binds to the ribosome during initial selection of tRNA, after binding of ternary complex but before GTP hydrolysis by EF-Tu. Viomycin binding renders the ribosome completely incapable of rejecting incorrect A-site bound tRNAs in both initial selection and proofreading. Viomycin sensitivity correlates with the accuracy of initial selection for the four different codon·anticodon pairs tested here. Our results demonstrate that, in contrast to current ideas about ‘induced-fit’, accuracy in initial selection is achieved primarily by increased dissociation rates for near-cognate tRNAs rather than by decreased rates of GTP hydrolysis. Further, our results imply that the ‘monitoring’ bases A1492 and A1493 rapidly fluctuate between active and inactive conformations when a near-cognate tRNA is present in the A site.

Place, publisher, year, edition, pages
Uppsala:
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-300234 (URN)
Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
4. Biochemical characterization of ΔTlyA mediated viomycin resistance
Open this publication in new window or tab >>Biochemical characterization of ΔTlyA mediated viomycin resistance
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-300235 (URN)
Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26

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