Antibiotics with novel modes of action targetingGram-negative bacteria are needed to resolve the antimicrobial resistance crisis 1-3 . These pathogens are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets 4,5 . The natural compound darobactin targets the insertase BamA 6 , the central unit of the essential BAM complex, which facilitates folding and insertion of outer membrane proteins 7-13 . BamA lacks a typical catalytic center, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here, we resolve the darobactin mode of action at the atomic level by a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two unique cyclizations pre-organize the darobactin peptide in a rigid βstrand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and open the path for rational design of antibiotics targeting this bacterial Achilles heel.The BAM complex was purified from E. coli outer membranes (OMs), reconstituted in n-dodecyl maltoside (DDM) micelles and incubated with darobactin A (darobactin). The cryo-EM reconstruction at 3.0 Å resolution revealed the position of a bound darobactin molecule (Fig. 1a, Extended Data Fig. 1, Supplementary Table 1). BamA features a lateral gate facing the membrane, formed by strands β1 and β16 through a kink in strand β16 at residue Gly807.Previous work showed that substrate-free BamA exists in two interchanging conformations with the gate either being open or being closed by the β16-strand straightening to zip up against

Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses, and kinetic modeling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudoreceiver domain Rec1, thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically invoke a downstream effect. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand and show that autophosphorylation is a reversible process.

The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin displayed up to tenfold higher potency than in synthetic membranes, highlighting the impact of native-like conditions in antibiotics development. The assay was successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of novel antibiotics targeting BAM.

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Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX–SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX–SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.