Ute Krengel

• Schmieder, Stefanie S; Cordara, Gabriele; Kersten, Flore; Steiner, Kevin; Samim, Clara H & Plaza, David F [Show all 13 contributors for this article] (2026). Structure and function of a fungal AB toxin-like chimerolectin involved in anti-nematode defense. EMBO Journal. ISSN 0261-4189. 45(13), p. 4786–4786. doi: 10.1038/s44318-026-00812-1. Full text in Research Archive Show summary

  Abstract 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 previously unknown family of fungal chimerolectins. Cryo-EM analysis to 3.2 Å resolution reveals five domains, with the four N-terminal β-trefoil fold (BTF) domains cradling a C-terminal domain, which exhibits an unusual α + β protein fold with some similarity to killer protein 4. Mutational analysis suggests that both terminal domains are functionally required for nematotoxicity. The first two BTF domains enable CCTX2 binding to glycosphingolipids with LacNAc or LacdiNAc glycoepitopes on nematode intestinal epithelial cells, whereas the biochemical function of the C-terminal domain remains unknown. Experiments in the model nematode Caenorhabditis elegans demonstrate that CCTX2 exploits the endocytic and retrograde trafficking machinery of cells in the intestinal epithelium to exert its toxicity and access the yet-to-be-identified intracellular target of the non-lectin domain. Our findings thus show that the molecular architecture and mode of action of CCTX2 is reminiscent of bacterial and plant AB toxins.

• Kersten, Flore; Cortés, Natalia Mojica; Serrano, Albert; Heim, Joel Benjamin; Guyette, Jessica L. & Cezar, Henrique Musseli [Show all 11 contributors for this article] (2026). A Single Amino Acid in the A2 Linker of Cholera Toxin Affects the Efficiency of Toxin Disassembly and Resulting Toxicity. ACS Omega. 11(38), p. 57763–57780. doi: 10.1021/acsomega.6c07022. Full text in Research Archive Show summary

  Abstract Cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are highly related AB5-type protein toxins that contain a catalytic A1 subunit, an A2 linker, and a cell-binding homopentamer of B subunits. The two proteins bind the same GM1 receptor and have similar levels of enzymatic activity toward their shared Gsα target, yet CT is more potent than LT. This difference has been mapped to a sequence of 11 amino acids (residues 226–236) near the C-terminus of the A2 linker. We now identify a single amino acid, aspartic acid at position 229, as a key contributor to the function of CT: substituting it for glutamic acid 229 from LT reduced the toxicity of CT to the level of LT. Toxin disassembly by protein disulfide isomerase (PDI), which is an essential event in the intoxication process, was less efficient for CTD229E and LT than for wild-type CT. This was further confirmed by a CT variant, in which four residues were converted into the LT sequence (D229E, I230V, T232I, and H233Y). To test our previous hypothesis that the positioning of the A1 subunit over the B pentamer critically determines toxin disassembly and thus potency, we determined crystal structures of both toxin variants to 1.6 Å resolution and calculated the angle at which the A2 linker exits the pore. The conformations of the two toxin chimeras lie between CT and LT, with the A2 C-terminus of the CT quadruple variant adopting essentially the same fold as LT. The A subunits of the toxin variants as well as that of LT showed a higher conformational freedom than CT as determined by molecular dynamics simulations, suggesting that a more rigid positioning of the A subunit drives toxin disassembly and potency. Our collective data uncover the critical role of D229 in toxin folding and structural dynamics, which in turn impacts toxin potency through effects on the efficiency of PDI-driven toxin disassembly.

• Nguyen, Adam; Heim, Joel Benjamin; Cordara, Gabriele; Chan, Matthew C.; Johannesen, Hedda & Charlesworth, Cristine [Show all 12 contributors for this article] (2026). Shared ligand-blocking mechanism but distinct conformational modulation by α5-targeting antibodies BIIG2 and MINT1526A. Structure. ISSN 0969-2126. doi: 10.1016/j.str.2026.08.001. Full text in Research Archive
• Sørensen, Henrik Vinther; Canals, Mateu Montserrat; Coder, Ayla Steffensen; Prévost, Sylvain; Krueger, Susan & Vaaje-Kolstad, Gustav [Show all 9 contributors for this article] (2026). Tangled Up in Fibers: How a Multidomain Lytic Polysaccharide Monooxygenase Binds Its Chitin Substrate. ACS Applied Materials & Interfaces. ISSN 1944-8244. doi: 10.1021/acsami.5c24418. Full text in Research Archive Show summary

  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. After determining the small-angle neutron scattering (SANS) contrast match point for chitin (47% D2O), we characterized the mesoscale structure of GbpA in complex with chitin by SANS and complemented the results with negative-stain electron microscopy. We found that GbpA binds rapidly to chitin, where it coats the chitin fibers and smooths their surface. In some locations, GbpA binding induces the formation of protein–chitin clumps containing a large number of GbpA molecules. Together, this suggests how the secretion of GbpA efficiently prepares the ground for microcolony formation by the bacteria.

• Petersson, Marcus; Pettersen, Jens Sivkær; Henriksen, Helena Bay; Duzs, Ágnes; Fernández-Quintero, Monica L. & Burlet, Nick Jean [Show all 15 contributors for this article] (2025). Attenuating ETEC virulence using a heat-labile enterotoxin-blocking binding protein. Gut microbes. ISSN 1949-0976. 18(1). doi: 10.1080/19490976.2025.2597567. Full text in Research Archive Show summary

  Bacterial enteric pathogens are major contributors to the global burden of diarrheal diseases and the associated consequences for human health including malnutrition, growth stunting, morbidity, and mortality. While mortality from diarrhea has decreased, incidence remains high, and better interventions for preventing disease are needed. Single-domain antibodies (i.e., VHHs), functioning as target-binding proteins in the gastrointestinal tract, have been proposed as a potential approach to mitigate bacterial pathogenesis. Here, we describe a mitigation strategy where precision binding of a bivalent VHH to the receptor-binding B-pentamer of heat-labile enterotoxin aggregates the AB5 toxin and impairs enterotoxigenic Escherichia coli colonization in a flow chamber model simulating the human intestine. The VHH construct also binds to the structurally similar cholera toxin and effectively abrogates its intestinal cell cytotoxicity in vitro. Based on these results, we believe that targeting virulence could emerge as a new strategy for the management of bacterial enteric pathogens, supporting gut health in at-risk populations alongside vaccination campaigns or in populations without access to vaccines.

• Canals, Mateu Montserrat; Cordara, Gabriele & Krengel, Ute (2025). Allostery. Quarterly Reviews of Biophysics. ISSN 0033-5835. 58(e5), p. 1–26. doi: 10.1017/S0033583524000209. Full text in Research Archive Show summary

  Allostery describes the ability of biological macromolecules to transmit signals spatially through the molecule from an allosteric site – a site that is distinct from orthosteric binding sites of primary, endogenous ligands – to the functional or active site. This review starts with a historical overview and a description of the classical example of allostery – hemoglobin – and other well-known examples (aspartate transcarbamoylase, Lac repressor, kinases, G-protein-coupled receptors, adenosine triphosphate synthase, and chaperonin). We then discuss fringe examples of allostery, including intrinsically disordered proteins and inter-enzyme allostery, and the influence of dynamics, entropy, and conformational ensembles and landscapes on allosteric mechanisms, to capture the essence of the field. Thereafter, we give an overview over central methods for investigating molecular mechanisms, covering experimental techniques as well as simulations and artificial intelligence (AI)-based methods. We conclude with a review of allostery-based drug discovery, with its challenges and opportunities: with the recent advent of AI-based methods, allosteric compounds are set to revolutionize drug discovery and medical treatments.

• Petersson, Marcus; Zingl, Franz G.; Rodriguez-Rodriguez, Everardo; Rendsvig, Jakob K. H.; Heinsøe, Heidi & Arendrup, Emma Wenzel [Show all 16 contributors for this article] (2025). Orally delivered toxin–binding protein protects against diarrhoea in a murine cholera model. Nature Communications. 16. doi: 10.1038/s41467-025-57945-w. Full text in Research Archive Show summary

  The ongoing seventh cholera pandemic, which began in 1961, poses an escalating threat to public health. There is a need for new cholera control measures, particularly ones that can be produced at low cost, for the one billion people living in cholera-endemic regions. Orally delivered VHHs, functioning as target-binding proteins, have been proposed as a potential approach to control gastrointestinal pathogens. Here, we describe the development of an orally deliverable bivalent VHH construct that binds to the B-pentamer of cholera toxin, showing that it inhibits toxin activity in a murine challenge model. Infant mice given the bivalent VHH prior to V. cholerae infection exhibit a significant reduction in cholera toxin–associated intestinal fluid secretion and diarrhoea. In addition, the bivalent VHH reduces V. cholerae colonization levels in the small intestine by a factor of 10. This cholera toxin–binding protein holds promise for protecting against severe diarrhoea associated with cholera.

• Heinzelbecker, Julia; Fauskanger, Marte; Jonson, Ida; Krengel, Ute; Løset, Geir Åge & Munthe, Ludvig Andre [Show all 7 contributors for this article] (2024). Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside. Frontiers in Immunology. 15:1331345, p. 1–12. doi: 10.3389/fimmu.2024.1331345. Full text in Research Archive Show summary

  Chimeric antigen receptor (CAR) T cell technology has ushered in a new era of immunotherapy, enabling the targeting of a broad range of surface antigens, surpassing the limitations of traditional T cell epitopes. Despite the wide range of non-protein tumor-associated antigens, the advancement in crafting CAR T cells for these targets has been limited. Owing to an evolutionary defect in the CMP-Neu5Ac hydroxylase (CMAH) that abolishes the synthesis of CMP-Neu5Gc from CMP-Neu5Ac, Neu5Gc is generally absent in human tissues. Despite this, Neu5Gc-containing antigens, including the ganglioside GM3(Neu5Gc) have consistently been observed on tumor cells across a variety of human malignancies. This restricted expression makes GM3(Neu5Gc) an appealing and highly specific target for immunotherapy. In this study, we designed and evaluated 14F7-28z CAR T cells, with a targeting unit derived from the GM3(Neu5Gc)-specific murine antibody 14F7. These cells exhibited exceptional specificity, proficiently targeting GM3(Neu5Gc)-expressing murine tumor cells in syngeneic mouse models, ranging from B cell malignancies to epithelial tumors, without compromising safety. Notably, human tumor cells enhanced with murine Cmah were effectively targeted and eliminated by the 14F7 CAR T cells. Nonetheless, despite the detectable presence of GM3(Neu5Gc) in unmodified human tumor xenografts, the levels were insufficient to trigger a tumoricidal T-cell response with the current CAR T cell configuration. Overall, our findings highlight the potential of targeting the GM3(Neu5Gc) ganglioside using CAR T cells across a variety of cancers and set the stage for the optimization of 14F7-based therapies for future human clinical application.

• Nguyen, Adam; Heim, Joel Benjamin; Cordara, Gabriele; Chan, Matthew C.; Johannesen, Hedda & Charlesworth, Christine [Show all 12 contributors for this article] (2024). Structural and functional characterization of integrin α5-targeting antibodies for anti-angiogenic therapy. BioRxiv. ISSN 0362-4331. doi: 10.1101/2025.01.08.631572. Full text in Research Archive
• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther; Kommedal, Eirik; Cordara, Gabriele & Vaaje-Kolstad, Gustav [Show all 7 contributors for this article] (2024). Calcium-binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environmental survival and infection. QRB Discovery. 5. doi: 10.1017/qrd.2024.14. Full text in Research Archive Show summary

  Despite major efforts toward its eradication, cholera remains a major health threat and economic burden in many low- and middle-income countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, for example, 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+ binding close to its carbohydrate-binding surface. In addition to the X-ray crystal structures of cation-LPMO complexes (to 1.5 Å resolution), we explored how the presence of ions affects the stability and activity of the protein. Calcium and magnesium ions were found to bind to GbpA specifically, with calcium ions – abundant in natural sources of chitin – having the strongest effect on protein stability. When the cation-binding site was rendered non-functional, a decrease in activity was observed, highlighting the importance of the structural elements stabilized by calcium. Our findings suggest a cation-binding site specific to GbpA and related LPMOs that may fine-tune binding and activity for its substrates during environmental survival and host infection.

• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther; Cordara, Gabriele; Vaaje-Kolstad, Gustav & Krengel, Ute (2023). Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environment survival and infection. BioRxiv. ISSN 0362-4331. doi: 10.1101/2023.12.22.573012. Full text in Research Archive
• Askarian, Fatemeh; Tsai, Chih-Ming; Cordara, Gabriele; Zurich, Raymond H.; Bjånes, Elisabet & Golten, Ole [Show all 22 contributors for this article] (2023). Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 120(30). doi: 10.1073/pnas.2301538120. Full text in Research Archive Show summary

  Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

• Cortés, Natalia Mojica; Kersten, Flore; Canals, Mateu Montserrat; III, G. Robb Huhn; Tislevoll, Abelone & Cordara, Gabriele [Show all 8 contributors for this article] (2023). Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration. Biochemistry. ISSN 0006-2960. doi: 10.1021/acs.biochem.3c00612. Full text in Research Archive Show summary

  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 emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax 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 perdeuteration 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 biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.

• Stocker, Christian; Khatanbaatar, Tamjidmaa; Bressan, Luca; Würth-Roderer, Kathrin; Cordara, Gabriele & Krengel, Ute [Show all 7 contributors for this article] (2023). Novel exported fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: Shikimate pathway enzymes teamed up in no man’s land. Journal of Biological Chemistry. ISSN 0021-9258. 299(10). doi: 10.1016/j.jbc.2023.105161. Full text in Research Archive Show summary

  Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in β-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, 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 pathway's end product Phe, and no catalytic benefit of the domain fusion compared with 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 Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the 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.

• Sørensen, Henrik Vinther; Canals, Mateu Montserrat; Loose, Jennifer Sarah Maria; Fisher, S. Zoë; Moulin, Martine & Blakeley, Matthew P. [Show all 9 contributors for this article] (2023). Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase. ACS Omega. 8(32), p. 29101–29112. doi: 10.1021/acsomega.3c02168. Full text in Research Archive Show summary

  Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

• Sørensen, Henrik Vinther; Montserrat-Canals, Mateu; Prévost, Sylvain François; Vaaje-Kolstad, Gustav; Bjerregaard-Andersen, Kaare & Lund, Reidar [Show all 7 contributors for this article] (2023). Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate. BioRxiv. ISSN 0362-4331. doi: 10.1101/2023.09.21.558757. Full text in Research Archive
• Thorbjørnsrud, Helen Vikdal; Bressan, Lucas; Khatanbaatar, Tamjidmaa; Carrer, Manuel; Würth-Roderer, Kathrin & Cordara, Gabriele [Show all 9 contributors for this article] (2023). What Drives Chorismate Mutase to Top Performance? Insights from a Combined In Silico and In Vitro Study. Biochemistry. ISSN 0006-2960. 62(3), p. 782–796. doi: 10.1021/acs.biochem.2c00635. Full text in Research Archive Show summary

  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 was 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 preorganized 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 molecular dynamics simulations and confirmed by kinetic investigations of engineered variants.

• Thorbjørnsrud, Helen Vikdal; Bressan, Luca; Khatanbaatar, Tamjidmaa; Carrer, Manuel; Würth-Roderer, Kathrin & Cordara, Gabriele [Show all 9 contributors for this article] (2022). What drives chorismate mutase to top performance? Insights from a combined in silico and in vitro study. BioRxiv. ISSN 0362-4331. doi: 10.1101/2022.11.08.515563. Full text in Research Archive
• Bousquet, Paula; Manna, Dipankar; Sandvik, Joe Alexander; Arntzen, Magnus Øverlie; Moreno, Ernesto & Sandvig, Kirsten [Show all 7 contributors for this article] (2022). SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7. Frontiers in Immunology. 13. doi: 10.3389/fimmu.2022.994790. Full text in Research Archive Show summary

  Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available via ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.

• Skåne, Anna; Edvardsen, Per Kristian Thorén; Cordara, Gabriele; Loose, Jennifer Sarah Maria; Leitl, Kira & Krengel, Ute [Show all 9 contributors for this article] (2022). Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis. BMC Microbiology. 22(1). doi: 10.1186/s12866-022-02590-2. Full text in Research Archive Show summary

  Background - Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context.<p> <p>Results - In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs.<p> <p>Conclusion - The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

• Serrano, Albert; Guyette, Jessica L.; Heim, Joel Benjamin; Taylor, Michael; Cherubin, Patrick & Krengel, Ute [Show all 8 contributors for this article] (2022). Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin. Scientific Reports. 12. doi: 10.1038/s41598-021-03939-9. Full text in Research Archive Show summary

  Abstract Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB 5 -type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT. The difference has been attributed to a more stable domain assembly for CT as compared to LT, but this explanation has not been directly tested and is arguable as toxin disassembly is an indispensable step in the cellular action of these toxins. We show here that PDI disassembles CT more efficiently than LT, which provides a possible explanation for the greater potency of the former toxin. Furthermore, direct examination of CT and LT domain assemblies found no difference in toxin stability. Using novel analytic geometry approaches, we provide a detailed characterization of the positioning of the A subunit with respect to the B pentamer and demonstrate significant differences in the interdomain architecture of CT and LT. Protein docking analysis further suggests that these global structural differences result in distinct modes of PDI-toxin interactions. Our results highlight previously overlooked structural differences between CT and LT that provide a new model for the PDI-assisted disassembly and differential potency of these toxins.

• Askarian, Fatemeh; Uchiyama, Satoshi; Masson, Helen; Sørensen, Henrik Vinther; Golten, Ole & Bunæs, Anne Cathrine [Show all 20 contributors for this article] (2021). The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection. Nature Communications. 12, p. 1–19. doi: 10.1038/s41467-021-21473-0. Full text in Research Archive Show summary

  The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of <i>Pseudomonas aeruginosa</i>, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by <i>P. aeruginosa</i>. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of <i>cbpD</i> rendered <i>P. aeruginosa</i> unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

• Bjerregaard-Andersen, Kaare; Abraha, Fana; Johannesen, Hedda; Oscarson, Stefan; Moreno, Ernesto & Krengel, Ute (2021). Key role of a structural water molecule for the specificity of 14F7 - An antitumor antibody targeting the NeuGc GM3 ganglioside. Glycobiology. ISSN 0959-6658. 31(11), p. 1500–1509. doi: 10.1093/glycob/cwab076. Full text in Research Archive Show summary

  Abstract Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody–antigen interactions.

• Manna, Dipankar; Cordara, Gabriele & Krengel, Ute (2020). Crystal structure of MOA in complex with a peptide fragment: A protease caught in flagranti. Current Research in Structural Biology. ISSN 2665-928X. doi: 10.1016/j.crstbi.2020.04.003. Full text in Research Archive Show summary

  The Marasmius oreades agglutinin (MOA) is the holotype of an emerging family of fungal chimerolectins and an active Ca2+/Mn2+-dependent protease, which exhibits a unique papain-like fold with special active site features. Here we investigated the functional significance of the structural elements differentiating MOA from other papain-like cysteine proteases. X-ray crystal structures of MOA co-crystallized with two synthetic substrates reveal cleaved peptides bound to the catalytic site, corresponding to the final products of the proteolytic reaction. Anomalous diffraction data on crystals grown in the presence of calcium and manganese, cadmium or zinc resolve the calcium/manganese preference of MOA and elucidate the inhibitory roles of zinc and cadmium towards papain-like cysteine proteases in general. The reported structures, together with activity data of MOA active site variants, point to a conservation of the general proteolysis mechanism established for papain. Ultimately, the findings suggest that papain and the papain-like domain of MOA are the product of convergent evolution.

• Bjerregaard-Andersen, Kaare; Johannesen, Hedda; Abraha, Fana; Šakanović, Aleksandra; Grosser, Daniel & Coskun, Ünal [Show all 11 contributors for this article] (2020). Insight into glycosphingolipid crypticity: Crystal structure of the anti-1tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.09.18.294777. Full text in Research Archive
• Fahrig-Kamarauskaitė, Jūratė; Würth-Roderer, Kathrin; Thorbjørnsrud, Helen Vikdal; Mailand, Susanne; Krengel, Ute & Kast, Peter (2020). Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance. Journal of Biological Chemistry. ISSN 0021-9258. 295(51), p. 17514–17534. doi: 10.1074/jbc.RA120.014924. Full text in Research Archive Show summary

  Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

• Heggelund, Julie Elisabeth; Heim, Joel Benjamin; Bajc, Gregor; Hodnik, Vesna; Anderluh, Gregor & Krengel, Ute (2019). Specificity of Escherichia coli heat-Labile enterotoxin investigated by single-site mutagenesis and crystallography. International Journal of Molecular Sciences. ISSN 1661-6596. 20(3). doi: 10.3390/ijms20030703. Full text in Research Archive Show summary

  Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

• Heim, Joel Benjamin; Hodnik, Vesna; Heggelund, Julie Elisabeth; Anderluh, Gregor & Krengel, Ute (2019). Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site. Scientific Reports. 9 . doi: 10.1038/s41598-019-48579-2. Full text in Research Archive Show summary

  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). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also L-fucose alone, 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. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called ‘secretors’.

• Burschowsky, Daniel; Thorbjørnsrud, Helen Vikdal; Heim, Joel Benjamin; Fahrig-Kamarauskaite, Jurate; Würth-Roderer, Kathrin & Kast, Peter [Show all 7 contributors for this article] (2018). Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp. Biochemistry. ISSN 0006-2960. 57(5), p. 557–573. doi: 10.1021/acs.biochem.7b01018. Full text in Research Archive Show summary

  Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM–CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis.

• Bousquet, Paula; Sandvik, Joe Alexander; Edin, Nina Frederike J & Krengel, Ute (2018). Hypothesis: Hypoxia induces de novo synthesis of NeuGc gangliosides in humans through CMAH domain substitute. Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 495(1), p. 1562–1566. doi: 10.1016/j.bbrc.2017.11.183. Full text in Research Archive Show summary

  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.

• Hayashi, Takahiro; Tinzl, Matthias; Mori, Takahiro; Krengel, Ute; proppe, jonny & Soetbeer, Janne [Show all 10 contributors for this article] (2018). Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme. Nature Catalysis. 1(8), p. 578–584. doi: 10.1038/s41929-018-0105-6. Full text in Research Archive
• Bjerregaard-Andersen, Kaare; Johannesen, Hedda; Abdel-Rahman, Noha Mansour Hassan; Heggelund, Julie Elisabeth; Hoås, Helene Mykland & Abraha, Fana [Show all 13 contributors for this article] (2018). Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour- specificity through an unusual H-chain CDR3 architecture. Scientific Reports. 8:10836, p. 1–11. doi: 10.1038/s41598-018-28918-5. Full text in Research Archive Show summary

  Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

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• Cortés, Natalia Mojica; Kersten, Flore; Serrano, Albert; Heim, Joel Benjamin; Cordara, Gabriele & Teter, Ken [Show all 8 contributors for this article] (2026). Why is cholera toxin more toxic than heat-labile enterotoxin? Insights from protein crystallography and MD simulations. Full text in Research Archive
• Krengel, Ute (2026). Allosteric regulation of chorismate mutase. Full text in Research Archive
• Krengel, Ute (2025). Protein-Carbohydrate Interactions in Infection Biology and Cancer. Full text in Research Archive
• Krengel, Ute (2025). Why is cholera more severe than traveler’s diarrhea? Full text in Research Archive
• Streitlein, Luca; Canals, Mateu Montserrat & Krengel, Ute (2024). Structural analysis of chorismate mutase variants and interaction with partner enzymes. Full text in Research Archive
• Albrechtova, Ema; Cordara, Gabriele & Krengel, Ute (2024). Structural characterisation of ATP-induced disassembly mechanisms in cytolethal distending toxin. Full text in Research Archive
• Cortés, Natalia Mojica; Kersten, Flore; Krengel, Ute; Teter, Ken; Cordara, Gabriele & Heim, Joel Benjamin [Show all 7 contributors for this article] (2024). Molecular insights into the differential toxicity of cholera toxin and heat-labile enterotoxin. Full text in Research Archive
• Cortés, Natalia Mojica; Kersten, Flore; Krengel, Ute; Cordara, Gabriele; Heim, Joel Benjamin & Teter, Ken [Show all 7 contributors for this article] (2024). Structural basis for the differential toxicity of cholera toxin and heat-labile enterotoxin. Full text in Research Archive
• Cortés, Natalia Mojica; Kersten, Flore; Krengel, Ute; Cordara, Gabriele; Serrano, Albert & Heim, Joel Benjamin [Show all 7 contributors for this article] (2024). Molecular insights into the differential toxicity of cholera toxin and heat-labile enterotoxin. Full text in Research Archive
• Crnic, Irene Fausk; Saragliadis, Athanasios; Cortés, Natalia Mojica; Kristiansen, Per Eugen & Krengel, Ute (2024). Molecular interactions between LPS and bacterial toxin. Full text in Research Archive
• Canals, Mateu Montserrat; Sekulic, Nikolina & Krengel, Ute (2024). UiO structural biology core facilities. Full text in Research Archive
• Kersten, Flore; Cordara, Gabriele & Krengel, Ute (2024). Cryo-EM structure of a fungal chimerolectin. Full text in Research Archive
• Sørensen, Henrik V.; Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare & Krengel, Ute (2024). New structural insights into bacterial colonization by Vibrio cholerae. Full text in Research Archive
• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther; Kommedal, Eirik; Cordara, Gabriele & Vaaje-Kolstad, Gustav [Show all 7 contributors for this article] (2024). Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environmental survival and infection                                               . Full text in Research Archive
• Stocker, Christian; Khatanbaatar, Tamjidmaa; Bressan, Luca; Würth-Roderer, Kathrin; Cordara, Gabriele & Krengel, Ute [Show all 7 contributors for this article] (2023). Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man's land. BioRxiv. ISSN 0362-4331. doi: 10.1101/2023.03.13.532365. Full text in Research Archive
• Kersten, Flore Marie Colette; Cordara, Gabriele & Krengel, Ute (2023). Cryo-EM reveals a novel domain fold of a fungal toxin. Full text in Research Archive
• Sekulic, Nikolina; Krengel, Ute & Brech, Andreas (2023). Minisymposium on CryoEM and CryoET: From knowledge to cures. Full text in Research Archive
• Krengel, Ute (2023). Protein-Carbohydrate Interactions in Infection and Cancer Biology. Full text in Research Archive
• Kersten, Flore Marie Colette; Cordara, Gabriele & Krengel, Ute (2022). Cryo-EM structure of a fungal chimerolectin. Full text in Research Archive
• Krengel, Ute (2022). Molecular Mechanisms of Cholera Pathogenesis. Full text in Research Archive
• Krengel, Ute (2022). Academia meets Industry. Full text in Research Archive
• Kersten, Flore Marie Colette; Cordara, Gabriele & Krengel, Ute (2022). Cryo-EM structure of a fungal toxin. Full text in Research Archive
• Kersten, Flore Marie Colette; Cordara, Gabriele & Krengel, Ute (2022). Cryo-EM structure of a fungal toxin. Full text in Research Archive
• Sekulic, Nikolina & Krengel, Ute (2022). PX-Oslo: Structural Biology Core Facilities. Full text in Research Archive
• Canals, Mateu Montserrat; Sørensen, Henrik Vinther; Oksanen, Esko & Krengel, Ute (2022). Neutron studies of the bacterial colonization factor GbpA. Full text in Research Archive
• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther & Krengel, Ute (2022). How salts modulate activity in chitin-active LPMOs relevant for environmental survival and human pahtogenicity. Full text in Research Archive
• Canals, Mateu Montserrat; Sørensen, Henrik Vinther; Oksanen, Esko O. & Krengel, Ute (2022). Structural studies on GbpA using neutron scattering methods. Full text in Research Archive
• Bousquet, Paula; Sandvik, Joe Alexander; Edin, Nina Frederike Jeppesen & Krengel, Ute (2022). Corrigendum to: Hypothesis: Hypoxia induces de novo synthesis of NeuGc gangliosides in humans through CMAH domain substitute (Biochemical and Biophysical Research Communications (2018) 495(1) (1562–1566), (S0006291X17323616), (10.1016/j.bbrc.2017.11.183)). Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 587, p. 166–166. doi: 10.1016/j.bbrc.2021.11.074. Full text in Research Archive
• Krengel, Ute (2021). My experience with HALOS: Making the most of structural biology. Full text in Research Archive
• Pesci, Giacomo; Johannesen, Hedda; Løset, Geir Åge & Krengel, Ute (2020). A promising antibody to treat cancer. How does it work? Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Løset, Geir Åge & Krengel, Ute (2020). Molecular basis of specificity of a promising anti-tumour antibody. Full text in Research Archive
• Cortez, Natalia Mojica; Sekulic, Nikolina & Krengel, Ute (2020). UiO Structural Biology Core Facilities: Who Are We and How Can We Help You? Full text in Research Archive
• Krengel, Ute (2020). UiO Structural Biology Core Facilities: Who Are We and How Can We Help You? Full text in Research Archive
• Cortez, Natalia Mojica & Krengel, Ute (2020). PX-Oslo- UiO Structural Biology Core Facilities. [Internet]. Department of Chemistry, UiO. Full text in Research Archive
• Sørensen, Henrik Vinther; Bjerregaard-Andersen, Kaare; Vaaje-Kolstad, Gustav; Krengel, Ute & Lund, Reidar (2020). Applying soft matter techniques to understand bacterial colonization. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Lund, Reidar; Browning, Kathryn & Bjerregaard-Andersen, Kaare (2019). Bacterial colonization investigated with X-ray and neutron techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Lund, Reidar & Krengel, Ute (2019). Applying X-rays and neutrons to understand a bacterial virulence factor. Full text in Research Archive
• Heggelund, Julie Elisabeth; Heim, Joel Benjamin & Krengel, Ute (2019). Cholera blood group association elucidated with synchrotron radiation. Full text in Research Archive
• Krengel, Ute (2019). Structural Chemistry - Past, Presence and Future. Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Ünal, Coskun; Løset, Geir Åge & Krengel, Ute (2019). Crystal structure of an anti-tumour antibody in complex with its tumour-specific antigen. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Vaaje-Kolstad, Gustav; Lund, Reidar; Browning, Kathryn & Bjerregaard-Andersen, Kaare [Show all 7 contributors for this article] (2019). Using X-rays and neutrons to understand a bacterial colonization factor. Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Heggelund, Julie Elisabeth; Løset, Geir Åge & Krengel, Ute (2019). 14F7 - A tumour specific antibody. Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2019). An anti-tumour antibody that specifically recognises N-glycolyl GM3. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Cardenas, Marite & Lund, Reidar [Show all 7 contributors for this article] (2019). Study of bacterial colonization with X-ray and neutron scattering techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Lund, Reidar & Krengel, Ute (2018). Borrowing material science techniques to study biological problems. Full text in Research Archive
• Krengel, Ute (2018). How to become an academic leader? Full text in Research Archive
• Krengel, Ute (2018). Cholera Toxin: Molecular Mechanisms and Drug Design. Full text in Research Archive
• Krengel, Ute (2018). Remote Control by Inter-Enzyme Allostery - Or: The Marvellous World of Chorismate Mutases. Full text in Research Archive
• Krengel, Ute (2018). Structure-Function Studies of Cholera Toxin: Molecular Underpinnings of Human Disease. Full text in Research Archive
• Krengel, Ute (2018). Interesting examples of allostery (?). Full text in Research Archive
• Johannesen, Hedda; hoås, Helene Mykland; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). 14F7, An anti-ganglioside scFv for cancer therapies. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn L.; Lund, Reidar; Krengel, Ute & Bjerregaard-Andersen, Kaare (2018). A surface active enzyme and colonization factor investigated by x-ray and neutron scattering. Full text in Research Archive
• Heim, Joel Benjamin; Teter, Kenneth & Krengel, Ute (2018). Towards the molecular basis for the differential toxicity of cholera toxin and heat-labile enterotoxin. Full text in Research Archive
• Johannesen, Hedda; Mykland, Helene Hoås; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). An anti-ganglioside scFv for cancer therapies. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Lund, Reidar; Bjerregaard-Andersen, Kaare & Krengel, Ute (2018). A surface active enzyme and colonization factor investigated by x-ray and neutron techniques. Full text in Research Archive
• Krengel, Ute (2018). How to design posters and write competitive grant proposals. Full text in Research Archive
• Khatanbaatar, Tamjidmaa & Krengel, Ute (2023). Partners in crime: Structure-function studies on chorismate mutases and their associated enzymes. Universitetet i Oslo. Full text in Research Archive Show summary

  Aromatic amino acids are essential building blocks for proteins. Humans lack enzymes that produce aromatic amino acids, thus they must consume them with their diet. In contrast, plants, bacteria and fungi are capable of de novo synthesis of aromatic amino acids, including the essential amino acids phenylalanine and tryptophan. Enzymes involved in the biosynthesis of aromatic amino acids are very promising drug targets, to fight bacterial and fungal infections or plant parasitosis. This PhD thesis is concerned with the study of chorismate mutase (CM), a fundamental metabolic enzyme. CM is often found associated to other enzymes from the same biosynthetic pathway, which regulate its activity. This work reports the structure of chorismate mutases from several bacteria, including human pathogens Mycobacterium tuberculosis and Pseudomonas aeruginosa, and novel bifunctional chorismate mutases from non-pathogenic bacteria. We shed light on their activity and regulation, including the mode of interaction with associated enzymes, laying the foundation for the discovery of new drugs.

• Krengel, Ute & Canals, Mateu Montserrat (2023). Molecular warfare: A structural biology view on pathogen weapons — GbpA from Vibrio cholerae — and host defenses — Vg from the honey bee. Universitetet i Oslo. Full text in Research Archive
• Stramrud, Mari & Krengel, Ute (2023). Molecular Interaction of Heat-Labile Enterotoxin B-pentamer with Lipopolysaccharides. Universitetet i Oslo. Full text in Research Archive Show summary

  Enterotoxigenic Escherichia coli (ETEC) forårsaker alvorlig diaré som på verdensbasis dreper millioner av mennesker hvert år. Bakteriens virulensfaktor er det varmelabile enterotoksinet (LT), nært beslektet til koleratoksinet (CT) som forårsaker kolera. LT er et AB5-holotoksin som består av en katalytisk aktiv A-subenhet plassert på toppen av en smultringformet B-pentamer (LTB), som er ansvarlig for å binde til epitelceller i tarmen. B-pentameren består av to bindingseter, det primære bindingssetet som binder til GM1-reseptoren på overflaten av epitelceller, og det sekundære bindingssetet som binder til blodgruppeantigener. I løpet av de siste årene har det blitt oppnådd økt kunnskap om det sekundære bindingssetet, og det ble oppdaget på hLTB isolert fra ETEC-stammer som infiserer mennesker. Tidligere studier har vist at hLTB binder til lipopolysakkarider (LPS) som finnes på den ytre membranen til bakterien, noe som forklarer forskjellen i leveringsmekanismer mellom CT og LT. LT er forankret til bakteriens overflate ved å binde seg til LPS og blir levert til vertscellen mens den er bundet til bakterielle membranvesikler, mens CT blir skilt ut i løselig form. Bindingskarakteristikkene til LPS-bindingssetet er fremdeles uklare, til tross for pågående forskning. Målet med dette prosjektet er å studere interaksjonen mellom LPS og hLTB ved bruk av NMR-spektroskopi. I dette arbeidet ble NMR-spektroskopi brukt til å undersøke interaksjonene mellom hLTB og LPS. I prosessen ble hLTB isotopmerket med 15N, produsert og renset for interaksjonsstudier. Tidligere eksperimenter som oppnådde et 1H-15N-TROSY NMR-spektrum av hLTB og titreringer av LPS som resulterte i signaltap ble gjentatt som kontroller i begynnelsen av prosjektet. Ytterligere titreringsforsøk ble utført med polymyksin B, LPS inkorporert i detergente miceller med DPC og hydrolysert LPS for å undersøke bindingskarakteristikkene og hvordan disse påvirket bindingen. Aminosyrene som deltar i interaksjonene mellom LPS og hLTB har blitt grundig studert. Resultatene støtter hypotesen om at residuene Q3 and F48 deltar både bindingen i til LPS and blodgruppe antigen. Fremgang er gjort i undersøkelsen av interaksjonen mellom LPS og hLTB, som i tillegg støtter teorien om et antatt tredje bindingssete og brøyter veiene for videre bindingsstudier.

• Falao, Eirik & Krengel, Ute (2023). Studies of interaction between the Vibrio cholerae colonization factor GbpA and chitin using NMR spectroscopy and electron microscopy imaging. Universitetet i Oslo. Full text in Research Archive Show summary

  The bacterium Vibrio cholerae is one of the most well-known contaminants of water and food sources across the world, and a cause of the disease cholera. It is particularly notorious for having outbreaks in underdeveloped parts of the world, or in areas affected by natural disasters or war, where access to clean drinking water is poor. Each year, cholera is estimated to cause 100 000 deaths. In addition to having the ability to colonize the human intestine, V. cholerae is known to colonize surfaces containing the polysaccharide chitin, found in the exoskeletons of several marine invertebrates. Colonization is believed to involve the adhesin N-acetyl glucosamine binding protein A (GbpA), which V. cholerae secretes into its aquatic environment. GbpA is a multifunctional protein with four domains, three of which have previously been structurally determined by x-ray crystallography. These are the N-terminal domain (D1), and the two middle domains (D2-3). D1 belongs to a class of enzymes called lytic polysaccharide monooxygenase (LPMO), which are known to cleave polysaccharides like chitin or cellulose. This domain is also known to interact with mucins, which are glycoproteins found in the human intestine. The functions of D2 and D3 are not known, but it has been hypothesized that they may play a role in anchoring V. cholerae to its substrate. The structure of the small C-terminal domain (D4) remains unknown, but it has been reported to also bind to chitinous surfaces. This thesis presents the results of interaction studies on GbpA with chitin, carried out by nuclear magnetic resonance (NMR) spectroscopy and electron microscope (EM) imaging. GbpA was produced using VmaxTM as an expression host. VmaxTM is a strain of Vibrio natriegens and a non-toxic relative of V. cholerae, possessing similar secretion pathways useful for GbpA expression and functionality. VmaxTM has previously been shown to express GbpA in high yields, which was confirmed in in this thesis. GbpA was also isotope-labelled with nitrogen-15 (15N) for NMR spectroscopy experiments. Although the NMR experiments yielded some inconclusive results, interactions between chitin and GbpA could be clearly observed in the EM-images obtained. Additionally, D4 was expressed alone with a glutathione S-transferase (GST) tag, using both Escherichia coli and VmaxTM as expression hosts. Although D4 was successfully isolated, extensive optimization of expression is required to acquire sufficient yields for v structure determination by NMR spectroscopy. An alternative method for isolating D4 was tested, where cleavage sites for TEV and HRV 3C proteases were inserted into the flexible linker separating D3 and D4, which would allow for GbpA to be expressed in high yields, from which D4 would be separated. Unfortunately, these cleavage sites could not be recognized by the proteases used, even though the same proteases could successfully cleave other proteins possessing the same cleavage sites. Predictions of the three-dimensional fold of D4 was made using AlphaFold and RoseTTAFold.

• Kristiansen, Helene Sørum; Krengel, Ute; Løset, Geir Åge & Progida, Cinzia (2022). Let it bind, and light you may find - Investigation of 14F7's cellular journey and effects. Universitetet i Oslo. Full text in Research Archive
• Hatlem, Daniel; Linke, Dirk & Krengel, Ute (2022). Probing the role of lipopolysaccharides in Gram-negative protein secretion and biogenesis. Universitetet i Oslo. Full text in Research Archive
• Cortes, Natalia Mojica & Krengel, Ute (2021). Catch me if you can - Interaction studies of AB5 toxins with PDI by small-angle scattering. Universitetet i Oslo. Full text in Research Archive
• Tislevoll, Abelone Martillas; Krengel, Ute & Sørensen, Henrik Vinther (2021). Isotope labelling and interaction studies of the Vibrio cholerae colonization factor GbpA. Universitetet i Oslo. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Lund, Reidar & Bjerregaard, Kaare (2021). Of Shellfish and Men: Applying X-ray and neutron techniques to investigate surface-active bacterial colonization factors. Universitetet i Oslo, Det matematisk-naturvitenskapelige fakultet. Full text in Research Archive Show summary

  Bacteria responsible for diseases such as cholera and pneumonia form biofilms for surviving in the environment and inside their human hosts. The process involves enzymes that target sugars, providing the bacteria with nutrients. An in-depth understanding of the mechanisms of these enzymes can potentially lead to new vaccines, but also new means to convert complex sugars to clean energy sources. We used X-ray and neutron scattering methods to elucidate how these enzymes bind to chitin in the environment and how they degrade it. The work reveals new insight into the structures and mechanisms of the enzymes and how the bacteria use them to survive and thrive in the environment and inside humans.

• Pesci, Giacomo; Krengel, Ute; Johannesen, Hedda & Løset, Geir Åge (2020). Follow the Light - Tracking 14F7’s cellular journey with luminescence. Universitetet i Oslo. Full text in Research Archive Show summary

  Cancer is one of the leading causes of premature death in the world and its incidence and mortality are rapidly growing worldwide. Despite the biological heterogeneity of cancer, most patients are still treated with generic therapies, which often result in negative aftermath on the patients’ health. Adverse effects can be reduced by treatments targeting molecules which are exclusively expressed by cancer cells. One such molecule is the ganglioside N-glycolyl GM3 neuraminic acid (NeuGc GM3), which is present on the surface of several human cancer cell types and is not found in healthy tissue. This tumor-specific ganglioside is targeted with high specificity by the monoclonal antibody 14F7, which was produced in Cuba two decades ago. Unlike most anti-tumor antibodies, 14F7 has the outstanding ability to induce cancer cell death without recruiting any component of the immune system. To date, the cell killing mechanism of 14F7 is still poorly understood. In order to fully exploit its potential in cancer therapy, the fate of 14F7 after binding to the target has to be unraveled. The aim of this Master’s project was to gain insights into the internalization abilities of 14F7. After binding to the ganglioside, does the antibody travel inside the cell? Here, a 14F7 single-chain variable fragment (scFv) was covalently connected to a luciferase called NanoLuc. Due to its bright luminescence and pH-dependent activity, the NanoLuc has been identified as a suitable reporter for internalization studies. The vector coding for the fusion protein 14F7 scFv-NanoLuc was cloned through a PCR- based method, named Gibson assembly. A production protocol based on control of the glucose feed in Escherichia coli was established. The protein was effectively purified with immobilized metal affinity chromatography followed by size-exclusion chromatography. The binding affinity of the 14F7 scFv domain to NeuGc GM3 was confirmed by ELISA. The NanoLuc revealed a very high brightness and a robust pH- dependent activity. A recently developed assay to quantify the amount of a cell surface receptor, named Topanga assay, allowed quantification of NeuGc GM3 on several cell lines. Using the Topanga assay as a starting point, a novel assay to obtain insights into the internalization kinetics was developed. Interestingly, high amounts of NeuGc GM3 on the cell surface corresponded to faster internalization. This work paves the way for the application of scFv-C1-Nluc in bioluminescence microscopy in live cells.

• Solbakke, Kristoffer Gudesen; Krengel, Ute; Johannesen, Hedda; Løset, Geir Åge & Bjerregaard-Andersen, Kaare (2019). 4G9: An antibody that potentially imitates a cancer-related carbohydrate. Universitetet i Oslo. Full text in Research Archive
• Thorbjørnsrud, Helen Vikdal; Krengel, Ute & Cascella, Michele (2018). Escaping Mediocrity - Activity and Regulation of a Catalytically Inefficient Enzyme. Universistetet i Oslo. Full text in Research Archive

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