Biology subjects

Krengel, U.

Publications and source records attributed to Krengel, U..

12 recordsLinked to original sources

bioRxiv2026

Conserved copper(I)-binding auxiliary domains link virulence-associated LPMOs to copper homeostasis in the intestinal environment

Multidomain lytic polysaccharide monooxygenases (LPMOs) are widely distributed in human pathogenic bacteria and increasingly recognized for promoting host interactions and virulence. Recent work has shown that immunizing mice with CbpD, a trimodular LPMO from Pseudomonas aeruginosa, provides protection against lethal P. aeruginosa infection. In several human intestinal pathogenic bacteria, LPMOs occur as tetra- and pentamodular proteins and their ability to interact with the host and facilitate virulence has primarily been ascribed to their ability to bind to N-acetylglucosamine containing glycans. Recently, AlphaFold predictions and substrate-binding studies of the Vibrio cholerae colonization factor GbpA suggested the presence of a copper-binding site in its non-catalytic third domain. In light of the recent demonstration that MUC2, the primary intestinal mucin, harbors two conserved and distinct copper binding sites for Cu(II) and Cu(I), we hypothesized that the auxiliary non-catalytic copper-binding domains of intestinal LPMOs evolved as a response to the intestinal environment. Here, we examine GbpA and homologous multidomain LPMOs from food-borne intestinal disease-causing Gram-positive bacteria from the genera Bacillus and Listeria. By combining mutagenesis with biochemical, spectroscopic, and computational approaches, we demonstrate that these multidomain LPMOs contain conserved, yet structurally distinct copper(I)-binding motifs on their non-catalytic third domain. A comprehensive review and reassessment of the literature spanning the past two decades reveals consistent links between the copper-binding ability of these LPMOs and host-pathogen interactions. Thus, our findings link these LPMOs to copper management in the intestine, offering an explanation for why their unique architecture is preserved across different bacterial lineages causing intestinal disease.

biochemistry↗

bioRxiv2025

Structure and function of a fungal AB toxin-like chimerolectin involved in anti-nematode defense

Fungal defense against predators largely relies on protein toxins, many of which are lectins. We previously showed that the production of the nematotoxin CCTX2 is upregulated in the Agaricomycete Coprinopsis cinerea upon predation by nematodes. Here, we classify CCTX2 as the founding member of a family of fungal chimerolectins. Cryo-EM analysis to 3.2 [A] resolution reveals five domains. The four N-terminal {beta}-trefoil fold (BTF) domains cradle a C-terminal domain, which exhibits a novel +{beta} protein fold. Mutational analysis shows that both N-terminal and C-terminal domains are required for nematotoxicity. While the biochemical function of the C-terminal domain remains unclear, the first two BTF domains enable CCTX2 to bind to glycosphingolipids with LacNAc or LacdiNAc glycoepitopes on nematode intestinal epithelial cells. Experiments in the model nematode Caenorhabditis elegans demonstrate that the chimerolectin CCTX2 exploits the endocytic and retrograde trafficking machinery of the target cell to exert its toxicity and obtain access to the yet-to-be-identified intracellular target of the non-lectin domain. The structure and mode of action of CCTX2 is reminiscent of bacterial and plant AB toxins.

microbiology↗

bioRxiv2025

Structural and functional characterization of integrin a5-targeting antibodies for anti-angiogenic therapy

Integrins are heterodimeric receptors important for cell adhesion and signaling. Integrin 5{beta}1 is a key mediator of angiogenesis and its dysregulation is associated with tumor progression and metastasis. Despite numerous efforts, 5{beta}1-targeting therapeutics have been unsuccessful due to poor efficacy and off-target effects. A contributing factor is our limited understanding of how integrin conformation influences interactions with therapeutics. Using cell-based functional assays, patient-derived xenografts, biophysics, and electron microscopy, we shed light on these relationships by characterizing two anti-5{beta}1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind 5{beta}1 with nanomolar affinity, reduce tube formation in vitro, and bind overlapping epitopes that block fibronectin binding. However, using electron microscopy, we reveal that while BIIG2 binding does not substantially alter the conformational states, MINT1526A preferentially recognizes the bent conformation and restricts the conformational ensemble. These insights can guide which aspects to prioritize to improve the design of future integrin-targeted therapeutics.

biochemistry↗

bioRxiv2023

Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environment survival and infection

Despite major efforts towards its eradication, cholera remains a major health and economic burden in many developing countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, e.g., on zooplankton. N-acetyl glucosamine binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca2+, Mg2+ or K+ to bind close to its carbohydrate-binding surface. In addition to the X-ray crystal structures, we explored how the presence of ions affects the stability of the protein, compared the new GbpA LPMO structures to those of other LPMOs, and discussed the relevance of our discovery for bacterial survival. Calcium ions, abundant in natural sources of chitin, have been found to have the strongest effect on GbpA stability. Our findings suggest a V. cholerae-specific cation-binding site in GbpA that may fine-tune activity and binding to the different substrates during environmental survival and host infection.

biochemistry↗

bioRxiv2023

Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration

Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (VmaxTM X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.

biochemistry↗

bioRxiv2023

Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that bind to and oxidize insoluble carbohydrate substrates, such as chitin or cellulose. This class of enzymes has attracted considerable attention due to their ability to convert biomaterials of high abundance into oligosaccharides that can be useful for producing biofuels and bioplastics. However, processes at the interface between solution and insoluble substrates represent a major challenge to biochemical and structural characterization. Here, we investigated the four-domain LPMO from Vibrio cholerae, N-acetyl glucosamine binding protein A (GbpA), to elucidate how it docks onto its insoluble substrate with its two terminal domains. First, we developed a protocol that allowed GbpA and chitin to form a stable complex in suspension, overcoming incompatibilities of the two binding partners with respect to pH. Using contrast variation small-angle neutron scattering (SANS), after determining the neutron scattering contrast match point for chitin (47% D2O), we characterized the structure of GbpA in complex with chitin by SANS, and by electron microscopy. We found that GbpA binds rapidly to chitin, where it spreads out on the chitin fibers, and smoothens their surface. In some locations, GbpA binding induces the formation of protein-chitin clumps containing hundreds of GbpA molecules. Together, this suggests how the secretion of GbpA efficiently prepares the ground for microcolony formation by the bacteria.

biochemistry↗

bioRxiv2023

Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man's land

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of phenylalanine. Surprisingly, exported CMs and CDTs exist in bacterial pathogens. Here, we report the discovery of novel and extremely rare exported bifunctional fusion enzymes, consisting of fused CM and CDT domains. Such enzymes were found in only nine bacterial species belonging to non-pathogenic {gamma}- or {beta}-proteobacteria. In {gamma}-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in {beta}-proteobacteria the order is inversed. The CM domains share 15-20% sequence identity with the AroQ{gamma} class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 {micro}M, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathways end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 [A] resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

biochemistry↗

bioRxiv2022

What drives chorismate mutase to top performance? Insights from a combined in silico and in vitro study

Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is pre-organized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by MD simulations and confirmed by kinetic investigations of engineered variants.

biochemistry↗

bioRxiv2020

Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is an anti-tumor antibody with high clinical potential, which has extraordinary specificity for NeuGc GM3, but does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 [A] crystal structure of the 14F7 binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Intriguingly, a water molecule appears to shape the specificity of 14F7. Using model membrane systems, we show that 14F7 recognizes NeuGc GM3 only above lipid concentrations that are likely to form glycolipid-rich domains. This "all-or-nothing" effect was exacerbated in giant unilamellar vesicles and multilamellar vesicles, whereas no binding was observed to 100 nm liposomes, emphasizing that the 14F7-NeuGc GM3 interaction is additionally modulated by membrane curvature. Unexpectedly, adding NeuAc GM3 strongly increased binding affinity to NeuGc GM3-containing liposomes. This effect may be important for tumor recognition, where the ubiquitous NeuAc GM3 may enhance 14F7 binding to NeuGc GM3-expressing cancer cells.

biochemistry↗

bioRxiv2018

Crystal structures reveal that Lewis-x and fucose bind to secondary cholera toxin binding site - in contrast to fucosyl-GM1

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). Recent studies suggest that the GM1 receptor may not be the only target of the CT, and that fucosylated receptors such as Lewisx (Lex) and histo-blood group antigens may also be important for cellular uptake and toxicity. However, where and how Lex binds to the CT remains unclear. Here we report the high-resolution crystal structure (1.5 [A]) of the receptor-binding B-subunit of the CT bound to the Lex trisaccharide, and present matching SPR data for CT holotoxins. Lex, and also O_SCPCAPLC_SCPCAP-fucose alone (at 500-fold molar excess), bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. The two binding sites are likely connected by allosteric cross-talk, potentially affecting toxin uptake. We also discuss why secretors are protected from severe cholera.\n\nAuthor summaryCholera is a severe diarrhoeal disease that is still a major killer in many parts of the world, especially in regions struck by natural disasters and wars. However, some individuals experience milder cholera symptoms. These so-called secretors, who have blood group antigens also in their bodily fluids like their saliva and the slimy mucus layer covering their stomach and intestines, appear to be somewhat protected. Here we present detailed atomic structures of cholera toxin and quantitative binding data that give clues of the protective mechanisms. Interactions of the protein toxin with sugar molecules are of crucial importance both for toxicity and protection. In addition, we identify a new tool for biochemical studies, and lay the groundwork for the design of cholera drugs and vaccines that may save countless human lives.

biochemistry↗

bioRxiv2017

Hypothesis: Hypoxia induces de novo synthesis of NeuGc gangliosides in humans

Immunotherapy is a growing field in cancer research. A privileged tumor-associated antigen that has received much attention is N-glycolyl (NeuGc) GM3. This ganglioside is present in several types of cancer, but is almost undetectable in human healthy tissues. However, its non-hydroxylated variant, NeuAc GM3, is abundant in all mammals. Due to a deletion in the human gene encoding the key enzyme for synthesis of NeuGc, humans, in contrast to other mammals, cannot synthesize NeuGc GM3. Therefore the presence of this ganglioside in human cancer cells represents an enigma. It has been shown that hypoxic conditions trigger the expression of NeuGc gangliosides, which not only serve as attractive targets for cancer therapy, but also as diagnostic and prognostic tumor marker. Here, we confirm hypoxia-induced expression of the NeuGc GM3 ganglioside also in HeLa cells and reveal several candidate proteins, in particular GM3 synthase and subunit B of respiratory complex II (SDHB), that may be involved in the generation of NeuGc GM3 by SILAC-based proteome analysis. These findings have the potential to significantly advance our understanding of how this enigmatic tumor-associated antigen is produced in humans, and also suggest a possible mechanism of action of anti-tumor antibodies that recognize hypoxia markers, such as 14F7.

cancer biology↗

bioRxiv2016

Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Atomic resolution crystal structures of these compounds in complexes with the cholera toxin give unprecedented atomic details of the molecular interactions, and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their relatively easy syntheses and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics.

biochemistry↗