Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.

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

TamA is an Escherichia coli Omp85 protein involved in autotransporter biogenesis. It comprises a 16-stranded transmembrane β-barrel and three POTRA domains. The 2.3-Å crystal structure reveals that the TamA barrel is closed at the extracellular face by a conserved lid loop. The C-terminal β-strand of the barrel forms an unusual inward kink, which weakens the lateral barrel wall and creates a gate for substrate access to the lipid bilayer.

regions of MAS are centrally connected by non-conserved linkers, which permit large-scale 10 relative motions in related systems 10 . To obtain a high-quality hybrid model, we divided MAS 11 into its condensing and modifying region, and excluded the flexibly tethered ACP (Fig. 1a). 12Three constructs of staggered C-terminal length were employed to define the length of 13 the condensing region (see Methods). All variants crystallized under the same condition; 14 structure determination mapped the last ordered residue to Glu887. The structure of the most 15 extended variant (1-892) was refined at 2.3 Å resolution (Extended Data Table 1a). MAS KS-AT 16 comprises an α/β-fold linker domain (LD) connecting AT to KS (Fig. 1b) Fig. 1a-c). Four interface segments of 6-19 amino acid (aa) length are disordered 8 (Fig. 1b), while equivalent regions are ordered in dimeric KS domains. connect to the modifying region, are proximal to the two-fold dimer axis above the KS active 15 site, as observed in previous condensing region structures 6,11,12 . 16The DHs connect the modifying region to the post-AT linkers of the condensing region. 17We solved crystal structures of a MAS DH construct (aa 884-1186), which overlaps in sequence 18 with the crystallized KS-AT, in two crystal forms with a total of six protomers arranged into 19 almost identical dimers (Extended Data Table 1a). The DH protomer is composed of two hot-dog 20 folds connected by a 20 aa hot-dog linker (Fig. 1c). A hydrophobic substrate binding tunnel 21 extends over both hot-dog folds with entrances near the C-terminus and at the distal end of hot-22 dog fold 2. Active site residues are contributed by both hot-dog folds and are located close to the Table 2a). In the DH dimer, the two protomers arrange 2 with their lateral ends bent towards the post-AT linkers with an interdomain angle of 222° 3 (Fig. 1c). The MAS DH dimer is distinct from the V-shaped DH arrangement in FAS 6 , which 4 lacks a dimerization interface and is bent into the opposite direction at an angle of 96°. MAS DH 5 rather resembles linear DH dimers of modPKSs with interdomain angles of 167-203°1 5-17 and a 6 common mode of dimerization via "handshake" interactions between β-strands of the N-terminal 7 hot-dog folds (Extended Data Fig. 1f-h). 8To obtain an authentic representation of the MAS modifying region, we crystallized in 9 presence of NADP + the complete DH-ΨKR-ER-KR segment, which is dimeric in solution based 10 on AUC. Based on SAXS, ACP deletion is not affecting the overall structure of this region 11 (Extended Data Fig. 2a-c). The crystallographic asymmetric unit reveals a complex packing of Fig. 3a, b) (aa 1948-1960) remains disordered in the absence of ligand, and 8 concomitantly, the nicotinamide moiety of NADP + is disordered (Extended Data Fig. 3d). The 9 MAS ΨKR exhibits an N-terminal β-α-β-α extension, which is commonly observed in modPKSs, 10but not in FASs 6,23 ; this extension exhibits increased flexibility as indicated by temperature 11 factor distributions (Extended Data ...

Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold.