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Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Wei Lin, Kalyan Das, David Degen, Abhishek Mazumder, Diego Duchi, Dongye Wang, View ORCID ProfileYon W. Ebright, Richard Y. Ebright, Elena Sineva, Matthew Gigliotti, Aashish Srivastava, Sukhendu Mandal, Yi Jiang, Yu Liu, Ruiheng Yin, Zhening Zhang, Edward T. Eng, Dennis Thomas, Stefano Donadio, Haibo Zhang, Changsheng Zhang, Achillefs N. Kapanidis, Richard H. Ebright
doi: https://doi.org/10.1101/237123
Wei Lin
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Kalyan Das
2Rega Institute and Department of Microbiology and Immunology, KU Leuven, 3000 Leuven, Belgium
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  • For correspondence: kalyan.das@kuleuven.be ebright@waksman.rutgers.edu
David Degen
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Abhishek Mazumder
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Diego Duchi
3Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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Dongye Wang
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Yon W. Ebright
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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  • ORCID record for Yon W. Ebright
Richard Y. Ebright
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Elena Sineva
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Matthew Gigliotti
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Aashish Srivastava
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Sukhendu Mandal
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Yi Jiang
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Yu Liu
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Ruiheng Yin
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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Zhening Zhang
4The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York NY 10027, USA
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Edward T. Eng
4The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York NY 10027, USA
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Dennis Thomas
5Center for Integrative Proteomics, Rutgers University, Piscataway NJ 08854, USA
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Stefano Donadio
6NAICONS Srl., 20139 Milan, Italy
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Haibo Zhang
7South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
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Changsheng Zhang
7South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
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Achillefs N. Kapanidis
3Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom
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Richard H. Ebright
1Waksman Institute and Department of Chemistry, Rutgers University, Piscataway NJ 08854, USA
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  • For correspondence: kalyan.das@kuleuven.be ebright@waksman.rutgers.edu
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Abstract

Fidaxomicin is an antibacterial drug in clinical use in treatment of Clostridium difficile diarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9 and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13 structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14 experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

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Posted December 20, 2017.
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Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)
Wei Lin, Kalyan Das, David Degen, Abhishek Mazumder, Diego Duchi, Dongye Wang, Yon W. Ebright, Richard Y. Ebright, Elena Sineva, Matthew Gigliotti, Aashish Srivastava, Sukhendu Mandal, Yi Jiang, Yu Liu, Ruiheng Yin, Zhening Zhang, Edward T. Eng, Dennis Thomas, Stefano Donadio, Haibo Zhang, Changsheng Zhang, Achillefs N. Kapanidis, Richard H. Ebright
bioRxiv 237123; doi: https://doi.org/10.1101/237123
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Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)
Wei Lin, Kalyan Das, David Degen, Abhishek Mazumder, Diego Duchi, Dongye Wang, Yon W. Ebright, Richard Y. Ebright, Elena Sineva, Matthew Gigliotti, Aashish Srivastava, Sukhendu Mandal, Yi Jiang, Yu Liu, Ruiheng Yin, Zhening Zhang, Edward T. Eng, Dennis Thomas, Stefano Donadio, Haibo Zhang, Changsheng Zhang, Achillefs N. Kapanidis, Richard H. Ebright
bioRxiv 237123; doi: https://doi.org/10.1101/237123

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