Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

Michael R. Wasserman; Arto Pulk; Zhou Zhou; Roger B. Altman; John C. Zinder; Keith D. Green; Sylvie Garneau-Tsodikova; Jamie H. Doudna Cate; Scott C. Blanchard

doi:10.1038/ncomms8896

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

Michael R. Wasserman
, Arto Pulk
, Zhou Zhou
, Roger B. Altman
, John C. Zinder
, Keith D. Green
, Sylvie Garneau-Tsodikova
, Jamie H. Doudna Cate
, Scott C. Blanchard

Research output: Contribution to journal › Article › peer-review

55 Scopus citations

Abstract

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin - paromomycin, ribostamycin and neamine - each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drugâ €™ s net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drugâ €™ s canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

Original language	English
Article number	7896
Journal	Nature Communications
Volume	6
DOIs	https://doi.org/10.1038/ncomms8896
State	Published - Jul 30 2015

Bibliographical note

Funding Information:
We thank R. Green (Johns Hopkins University) for providing the S13 knockout strain, P. Schultz for providing the L5 knockout strain (Scripps Research Institute), T. Suzuki (University of Tokyo) for providing the pKK3535 ribosome plasmids, D. Crish (Wayne State University) for providing 6′-deoxy-paromomycin and M. O’Connor (University of Missouri-Kansas City) for helpful discussions throughout the course of this work. We also acknowledge helpful discussions and insights provided by all members of the Blanchard and Cate laboratories and J. Headd (Lawrence Berkeley National Laboratory) for help with PHENIX refinement. This work was supported by the US National Institutes of Health (2R01GM079238 to S.C.B., 1R01GM65050 to J.H.D.C., 1R01AI090048 to S.G.-T. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory), the Human Frontiers in Science Program (RGY0088), the National Science Foundation (0644129) and the US Department of Energy (DE-AC0376SF00098 for the SIBYLS and 8.3.1 beamlines at the ALS, Lawrence Berkeley National Laboratory).

Funding

We thank R. Green (Johns Hopkins University) for providing the S13 knockout strain, P. Schultz for providing the L5 knockout strain (Scripps Research Institute), T. Suzuki (University of Tokyo) for providing the pKK3535 ribosome plasmids, D. Crish (Wayne State University) for providing 6′-deoxy-paromomycin and M. O’Connor (University of Missouri-Kansas City) for helpful discussions throughout the course of this work. We also acknowledge helpful discussions and insights provided by all members of the Blanchard and Cate laboratories and J. Headd (Lawrence Berkeley National Laboratory) for help with PHENIX refinement. This work was supported by the US National Institutes of Health (2R01GM079238 to S.C.B., 1R01GM65050 to J.H.D.C., 1R01AI090048 to S.G.-T. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory), the Human Frontiers in Science Program (RGY0088), the National Science Foundation (0644129) and the US Department of Energy (DE-AC0376SF00098 for the SIBYLS and 8.3.1 beamlines at the ALS, Lawrence Berkeley National Laboratory).

Funders	Funder number
Searle, Human Frontiers Science Program	RGY0088
US Department of Energy	DE-AC0376SF00098
US National Institutes for Health	1R01AI090048, 2R01GM079238, 1R01GM65050
National Science Foundation Arctic Social Science Program	0644129
National Childhood Cancer Registry – National Cancer Institute	CA92584
National Institute of Allergy and Infectious F32-AI286447 Cydney N. Johnson Diseases National Institute of Allergy and Infectious R01AI168214 Jason W. Rosch Diseases National Institute of Allergy and Infectious P30 Cydney N. Johnson Diseases National Institute of Allergy and Infectious R00-AI166116 Christopher D. Radka Diseases National Institute of Allergy and Infectious T32-AI106700 Cydney N. Johnson Diseases National Institute of Allergy and Infectious R01AI192221 Jason W. Rosch Diseases National Inst...	R01AI090048
Lawrence Berkeley National Laboratory

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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10.1038/ncomms8896

Cite this

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title = "Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions",

abstract = "Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin - paromomycin, ribostamycin and neamine - each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug{\^a} €{\texttrademark} s net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug{\^a} €{\texttrademark} s canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.",

author = "Wasserman, \{Michael R.\} and Arto Pulk and Zhou Zhou and Altman, \{Roger B.\} and Zinder, \{John C.\} and Green, \{Keith D.\} and Sylvie Garneau-Tsodikova and \{Doudna Cate\}, \{Jamie H.\} and Blanchard, \{Scott C.\}",

note = "Funding Information: We thank R. Green (Johns Hopkins University) for providing the S13 knockout strain, P. Schultz for providing the L5 knockout strain (Scripps Research Institute), T. Suzuki (University of Tokyo) for providing the pKK3535 ribosome plasmids, D. Crish (Wayne State University) for providing 6′-deoxy-paromomycin and M. O{\textquoteright}Connor (University of Missouri-Kansas City) for helpful discussions throughout the course of this work. We also acknowledge helpful discussions and insights provided by all members of the Blanchard and Cate laboratories and J. Headd (Lawrence Berkeley National Laboratory) for help with PHENIX refinement. This work was supported by the US National Institutes of Health (2R01GM079238 to S.C.B., 1R01GM65050 to J.H.D.C., 1R01AI090048 to S.G.-T. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory), the Human Frontiers in Science Program (RGY0088), the National Science Foundation (0644129) and the US Department of Energy (DE-AC0376SF00098 for the SIBYLS and 8.3.1 beamlines at the ALS, Lawrence Berkeley National Laboratory).",

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doi = "10.1038/ncomms8896",

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T1 - Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

AU - Wasserman, Michael R.

AU - Pulk, Arto

AU - Zhou, Zhou

AU - Altman, Roger B.

AU - Zinder, John C.

AU - Green, Keith D.

AU - Garneau-Tsodikova, Sylvie

AU - Doudna Cate, Jamie H.

AU - Blanchard, Scott C.

N1 - Funding Information: We thank R. Green (Johns Hopkins University) for providing the S13 knockout strain, P. Schultz for providing the L5 knockout strain (Scripps Research Institute), T. Suzuki (University of Tokyo) for providing the pKK3535 ribosome plasmids, D. Crish (Wayne State University) for providing 6′-deoxy-paromomycin and M. O’Connor (University of Missouri-Kansas City) for helpful discussions throughout the course of this work. We also acknowledge helpful discussions and insights provided by all members of the Blanchard and Cate laboratories and J. Headd (Lawrence Berkeley National Laboratory) for help with PHENIX refinement. This work was supported by the US National Institutes of Health (2R01GM079238 to S.C.B., 1R01GM65050 to J.H.D.C., 1R01AI090048 to S.G.-T. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory), the Human Frontiers in Science Program (RGY0088), the National Science Foundation (0644129) and the US Department of Energy (DE-AC0376SF00098 for the SIBYLS and 8.3.1 beamlines at the ALS, Lawrence Berkeley National Laboratory).

PY - 2015/7/30

Y1 - 2015/7/30

N2 - Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin - paromomycin, ribostamycin and neamine - each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drugâ €™ s net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drugâ €™ s canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

AB - Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin - paromomycin, ribostamycin and neamine - each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drugâ €™ s net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drugâ €™ s canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

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AN - SCOPUS:84938343295

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JO - Nature Communications

JF - Nature Communications

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ER -

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

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