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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. Catch bonds have a role in bacterial adhesion and infection by uropathogenic E. coli. Here, the authors report crystal structures, molecular dynamics simulations, ligand binding analysis and cell tracking to characterise the catch bond interaction between the adhesin FimH and carbohydrate receptors.

A hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase, a multienzyme involved in the biosynthesis of mycobacterial branched-chain fatty acids, exemplifies the organization of fully-reducing polyketide synthases. A polyketide synthase structure revealed Mycocerosic acid synthase (MAS) is a mycobacterial polyketide synthase (PKS) responsible for the biosynthesis of C 20 –C 28 branched-chain fatty acids, key components of the specialized mycobacterial cell wall. These authors report a hybrid crystal structure of MAS from Mycobacterium smegmatis . Many commonly used drugs are either naturally occurring polyketides or their derivatives. This work represents the first high-quality model of a full PKS module, and it could be used to help dissect and re-engineer other PKSs to make new, non-natural polyketides. Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities 1 , 2 , 3 . Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line 3 , whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs) 3 . However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment 4 , 5 . PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases 6 . Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like 7 , 8 PKSs. It is involved in the biosynthesis of C 20 –C 28 branched-chain fatty acids, which are important virulence factors of mycobacteria 9 . Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts. Urease is a nickel enzyme responsible for catalyzing the conversion of urea into ammonia and carbon dioxide. Here the authors report a high resolution cryo-EM structure of urease from the bacterial pathogen Yersinia enterocolitica , providing a detailed visualization of the urease bimetal active site and a basis for drug development.

The replacement of ionizable functional groups that are predominantly charged at physiological pH with neutral bioisosteres is a common strategy in medicinal chemistry; however, its impact on binding affinity is often context-dependent. Here, we investigated a series of amide derivatives of a glycomimetic E-selectin ligand, in which the carboxylate group of the lead compound is substituted with a range of amide and isosteric analogs. Despite the expected loss of the salt-bridge interaction with Arg97, several amides retained or even improved the binding affinity. Co-crystal structures revealed conserved binding poses across the series, with consistent interactions involving the carbonyl oxygen of the amide and the key residues Tyr48 and Arg97. High-level quantum chemical calculations ruled out a direct correlation between carbonyl partial charges and affinity. Instead, a moderate correlation was observed between ligand binding and the out-of-plane pyramidality of the amide nitrogen, suggesting a favorable steric adaptation within the binding site. Molecular dynamics (MD) simulations revealed that high-affinity ligands exhibit enhanced solution-phase pre-organization toward the bioactive conformation, likely reducing the entropic penalty upon binding. Further analysis of protein-ligand complexes using Molecular mechanics/Generalized born surface area (MM-GB/SA) decomposition suggested minor lipophilic contributions from amide substituents. Taken together, this work underscores the importance of geometric and conformational descriptors, beyond classical electrostatics, in driving affinity in glycomimetic ligand design and provides new insights into the nuanced role of amides as carboxylate isosteres in protein-ligand recognition.

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/–condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

The TMEM175 family constitutes recently discovered K+channels that are important for autophagosome turnover and lysosomal pH regulation and are associated with the early onset of Parkinson Disease. TMEM175 channels lack a P-loop selectivity filter, a hallmark of all known K+ channels, raising the question how selectivity is achieved. Here, we report the X-ray structure of a closed bacterial TMEM175 channel in complex with a nanobody fusion-protein disclosing bound K+ ions. Our analysis revealed that a highly conserved layer of threonine residues in the pore conveys a basal K+ selectivity. An additional layer comprising two serines in human TMEM175 increases selectivity further and renders this channel sensitive to 4-aminopyridine and Zn2+. Our findings suggest that large hydrophobic side chains occlude the pore, forming a physical gate, and that channel opening by iris-like motions simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus , a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development. Computational search identifies dynobactin A which is a systemically active, natural-product peptide antibiotic that kills Gram-negative bacteria.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Prolyl cis/trans isomerization is a rate-limiting step in protein folding, often coupling directly to the acquisition of native structure. Here, we investigated the interplay between folding and prolyl isomerization in the N2 domain of the gene-3-protein from filamentous phage fd, which adopts a native-state cis/trans equilibrium at Pro161. Using mutational and Φ-value analysis, we identified a discrete folding nucleus encompassing the β-strands surrounding Pro161. These native-like interactions form early in the folding pathway and provide the energy to shift the cis/trans equilibrium toward the cis form. Variations distant from the Pro161-loop have minimal impact on the cis/trans ratio, underscoring the spatial specificity and localized control of the isomerization process. Using NMR spectroscopy, we determined the structures for both native N2 forms. The cis- and trans-Pro161 conformations are overall identical and exhibit only slight differences around the Pro161-loop. The cis-conformation adopts a more compact structure with improved backbone hydrogen bonding, explaining the approximately 10 kJ·mol−1 stability increase of the cis state. Our findings highlight that prolyl isomerization in the N2 domain is governed by a localized folding nucleus rather than global stability changes. This localized energetic coupling ensures that proline isomerization is not simply a passive, slow step but an integral component of the folding landscape, optimizing both the formation of native structure and the establishment of the cis-conformation.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19845-z.