ABSTRACT
Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM 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." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , DNA-Directed RNA Polymerases/antagonists & inhibitors , Escherichia coli/drug effects , Fidaxomicin/pharmacology , Mycobacterium tuberculosis/drug effects , Transcription, Genetic/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Binding Sites , Cryoelectron Microscopy , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/ultrastructure , Drug Design , Drug Resistance, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/ultrastructure , Fidaxomicin/chemistry , Fidaxomicin/metabolism , Fluorescence Resonance Energy Transfer , Gene Expression Regulation, Bacterial/drug effects , Models, Molecular , Mutation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/ultrastructure , Protein Binding , Protein Conformation , Single Molecule Imaging , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Structure-Activity RelationshipABSTRACT
Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.
