Structural basis for the unexpected activity of rifamycin B against rifampicin-resistant RNA polymerase

Abstract

Rifamycins inhibit bacterial transcription by targeting RNA polymerase (RNAP), but their clinical effectiveness is limited by the rapid emergence of resistance caused by mutations within the rifamycin-binding pocket. Rifamycin B (Rif B), one of the earliest discovered members of this antibiotic family and a precursor of clinically used derivatives, has remained poorly characterized because of its chemical instability and relatively weak antibacterial activity. Here, we revisit Rif B using biochemical and structural approaches. We show that Rif B remains sufficiently stable under assay conditions and retains inhibitory activity against RNAP variants carrying clinically relevant rifampicin-resistance mutations. We report the first crystal structure of Rif B and determine the structure of Rif B bound to bacterial RNAP. The structures reveal that the distinctive C-4 O-carboxymethyl substituent of Rif B forms an intramolecular interaction in the free molecule but establishes a salt bridge with fork loop 2 of the RNAP {beta}-subunit upon binding. This additional interaction explains the reduced sensitivity of Rif B to resistance-associated substitutions and identifies the C-4 position as an underexplored site for rational rifamycin modification. These findings redefine Rif B as a mechanistically distinct rifamycin scaffold and provide new insights for developing inhibitors targeting rifampicin-resistant RNAP.