The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
The darobactins are promising new antibiotics that inhibit the BAM complex, an essential membrane‐protein foldase in Gram‐negative bacteria. Here, we use EPR spectroscopy (PELDOR, or DEER) distance measurements to monitor the effect of darobactin B on BAM in E. coli cells, showing it stabilises BAM in a lateral gate closed conformational state. Our results provide the first direct evidence for conformational selection by a darobactin in the cellular context.
Keywords: BAM; Cryoem; Darobactin; In-Cell EPR; PELDOR.
© 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.