Darobactin B Stabilises a Lateral‐Closed Conformation of the BAM Complex in E. coli Cells

Abstract

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.

Lateral gating mechanism and plasticity of the BAM complex in micelles andE. coli

“…Thus, our observations also provide an explanation for the essentiality of BamD for the function of WT BamA, which also might interact with the substrate (70,71). The exact role for the other lipoproteins remains to be elucidated and they might be required to facilitate and or accelerate the folding of diverse substrates in the physiological environments (5,54,72,73). The possibility to observe BAM in E. coli and in the native outer membranes (35,74,75) provides a great opportunity to elucidate the dynamic basis of BAM function as well as its inhibition by novel compounds in the native asymmetric bilayers.…”

ATP‐independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next‐generation therapeutics

“…It binds to the BamA lateral gate and stabilizes it, preventing nOMP biogenesis. Its homolog darobactin B (j) arrests the BAM complex in the “lateral closed” conformation (Haysom et al, 2023). (k) Dynobactins, also derived from distantly related biosynthetic gene clusters of darobactins, have similar structure and function, but superior activity and desirable inhibitory properties (Miller et al, 2022).…”

“…With increasing availability of elegant instrumentation, more compounds and isoforms are now being tested. Darobactin B (DarB; homolog of darobactin A; Figure 6j) has also been investigated recently for its BAM complex binding properties (Haysom et al, 2023). Double electron–electron resonance (DEER) spectroscopy combined with site‐directed spin labelling (SDSL) and cryoEM studies in vitro and in vivo have shown that DarB bears functional conservation of darobactin, and binds to the lateral gate of BamA keeping it in a lateral closed state.…”

“…Given the resurgence of drug‐resistant Gram‐negative bacterial pathogens, the BAM complex is being actively examined as a novel therapeutic target for inhibitor design and screens of peptidotherapeutics (Di Somma et al, 2022; Diederichs et al, 2021; Doyle & Bernstein, 2022; Ghequire et al, 2018; Hagan et al, 2015; Hart et al, 2019; Imai et al, 2019; Li et al, 2020; Luther et al, 2019; Miller et al, 2022; Overly Cottom et al, 2023; Steenhuis, van Ulsen, et al, 2021; Storek et al, 2018; Tomasek & Kahne, 2021; Urfer et al, 2016; Xu et al, 2023). Most recent advances in the identification and design of inhibitors that specifically target this essential bacterial protein (Haysom et al, 2023; Miller et al, 2022; Overly Cottom et al, 2023; Seyfert et al, 2023; Steenhuis, van Ulsen, et al, 2021; Xu et al, 2023) now offer promise in successfully developing translational therapeutics for drug‐resistant bacterial pathogens. This review presents the structures of the BAM complex and its interactome, outlines known modes of BAM complex function, and describes recent successes in the identification and characterization of BAM‐specific inhibitors as next‐generation therapeutics.…”

New Genetically Engineered Derivatives of Antibacterial Darobactins Underpin Their Potential for Antibiotic Development

“…In the coming years, increasing antimicrobial resistance (AMR) is expected to induce a rise in the number of deaths of the growing global population from roughly 1.3 million to up to 10 million annually. − In particular, resistant pathogens appear in clinics more often, among other factors in combination with nosocomial infections, leading to increased mortality rates. − Most new antibiotics reaching the development stage are derivatives of known chemical classes with a known target and mode of binding (MoB), resulting in potentially rapid development of cross-resistance. − Thus, the search for antibacterial compounds acting on innovative target sites is urgently required. The recently discovered darobactins are a novel class of antibiotics showing promise for meeting that demand. , Native darobactins found in Photorhabdus khanii are ribosomally produced and post-translationally modified peptides (RiPPs). , They selectively target the outer membrane protein (OMP) BamA, the major component of the BamABCDE (BAM) complex, and inhibit the insertion and folding of OMPs, ultimately resulting in cell lysis. − The target site of darobactins is not addressed by commercially available antibiotics. Consequently, the risk of cross-resistance is lower, and they are proven to exhibit an auspicious broad-spectrum Gram-negative activity against clinically relevant and multiresistant pathogens such as Pseudomonas aeruginosa (P.…”