Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin

Abstract

Significance Many bacteria produce antimicrobial peptides for survival under stressful conditions. Some of these antimicrobial peptides are lasso peptides, which have a unique lasso-like topology and have generated great interest as a result of their stability in harsh conditions and amenability to functional engineering. In this study, we determined crystal structures of two lasso peptides, microcin J25 and capistruin, bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). The structures define peptide inhibitor–RNAP interactions that are important for inhibition and provide detailed insight into how the peptides inhibit RNAP function. This work provides a structural basis to guide the design of more potent lasso peptide antimicrobial approaches. We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739–751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.