PT - JOURNAL ARTICLE AU - Wei Lin AU - Kalyan Das AU - David Degen AU - Abhishek Mazumder AU - Diego Duchi AU - Dongye Wang AU - Yon W. Ebright AU - Richard Y. Ebright AU - Elena Sineva AU - Matthew Gigliotti AU - Aashish Srivastava AU - Sukhendu Mandal AU - Yi Jiang AU - Yu Liu AU - Ruiheng Yin AU - Zhening Zhang AU - Edward T. Eng AU - Dennis Thomas AU - Stefano Donadio AU - Haibo Zhang AU - Changsheng Zhang AU - Achillefs N. Kapanidis AU - Richard H. Ebright TI - Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3) AID - 10.1101/237123 DP - 2017 Jan 01 TA - bioRxiv PG - 237123 4099 - http://biorxiv.org/content/early/2017/12/20/237123.short 4100 - http://biorxiv.org/content/early/2017/12/20/237123.full AB - 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.