The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase

Abstract

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

Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering

“…Darobactin is a promising candidate for biosynthetic structure engineering attempts due to its excellent antibacterial activity profile, the hypothesised natural variability in its amino acid composition, 15 and its presumed insensitivity towards amino acid mutations in its binding site. 18 Thus, we investigated the plasticity of our heterologous production system aiming at the production of novel darobactin derivatives with improved antibacterial properties. To this end, we designed four DNA fragments in silico including the entire darA gene with an altered core sequence encoding the propeptides of the hypothetical natural darobactins B–E ( Table 1 and Fig.…”

“…It was recently shown that darobactin A binds to the open form BamA lateral gate with high affinity by mimicking a β-strand in its BamA-bound binding pose. 18 Consequently, the β-signal binding site of BamA is blocked, cognate substrate binding is inhibited and nascent OM proteins are not inserted into the OM. Darobactin A is a bicyclic heptapeptide and part of the RiPP secondary metabolite class characterized by the precursor peptide amino acid sequence W 1 N 2 W 3 S 4 K 5 S 6 F 7 ( Fig.…”

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

“…Overall, the structure suggests that BAM binds to β-signals to correctly orient the C-terminal strands of new OMPs into the OM during the folding process. Interestingly, darobactin (a recently discovered BAM inhibitor) (Imai et al, 2019) and the β-signal interact with BamAβ1 in a highly similar way; like EspP F1300, the terminal phenylalanine of the darobactin peptide is positioned over BamA G424 (Kaur et al, 2021) (Figure 1G). Therefore, our structure not only provides the structural basis for native OMP β-signal binding by BAM during assembly, but also definitively shows that darobactin is a competitive inhibitor of OMP substrate recognition and thereby helps to explain its bactericidal potency.…”

“…Presumably the mutations reduce the binding affinity of incoming OMPs to BAM, prevent their progression to the hybrid-barrel stage, and result in exposure to periplasmic proteases. Our discovery of this binding site may also enable the design of novel competitive inhibitors of β-signal binding and the further development of lead compounds such as darobactin (Imai et al, 2019; Kaur et al, 2021) that act as potent antibiotics against multidrug-resistant Gram-negative pathogens.…”

“…The structural dynamics that occur as OMPs transition from an incompletely folded state to a fully folded β-barrel remain unclear. However, available evidence suggests that OMP β-signals may be recognized by BAM and that the unusual conformational malleability of BAM (particularly BamA) may facilitate the folding process (Doerner and Sousa, 2017; Doyle and Bernstein, 2019; Hagan et al, 2015; Iadanza et al, 2016; Kaur et al, 2021; Lundquist et al, 2018; Noinaj et al, 2014; Tomasek et al, 2020; White et al, 2021). Interestingly, BamA does not contain a canonical β-signal at its C-terminus, but instead has a “kinked” structure that causes its terminal residues to move dynamically and generate a unique unstable β-seam (that forms hydrogen-bonds poorly) (Lundquist et al, 2018; Noinaj et al, 2013).…”