Publications
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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.
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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.
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Canals, Mateu Montserrat; Schnelle, Kilian; Leipart, Vilde; Halskau, Øyvind; Amdam, Gro Vang & Moeller, Arne
[Show all 8 contributors for this article]
(2025).
Cryo-EM structure of native honey bee vitellogenin.
Nature Communications.
16(1).
doi:
10.1038/s41467-025-58575-y.
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Vitellogenin (Vg) is the main yolk precursor lipoprotein in almost all egg-laying animals. In addition, along its evolutionary history, Vg has developed a range of new functions in different taxa. In the honey bee, Vg has functions related to immunity, antioxidant protection, social behavior and longevity. However, the molecular mechanisms underlying Vg functionalities are still poorly understood. Here, we report the cryo-EM structure of full-length honey bee Vg, one-step purified directly from hemolymph. The structure provides structural insights into the overall domain architecture, including the lipid binding cavity and the previously uncharacterized von Willebrand factor type D domain. A domain of unknown function has been identified as a C-terminal cystine knot domain based on structural homology. Information about post-translational modifications, cleavage products, metal and lipid binding allow an improved understanding of the mechanisms underlying the range of Vg functionalities. The findings have numerous implications for the structure-function relationship of vitellogenins of other species as well as members of the same protein superfamily, which share the same structural elements.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Leipart, Vilde; Canals, Mateu Montserrat; Cunha, Eva Mariana de Sousa; Luecke, Hartmut; Herrero-Galán, Elías & Halskau, Øyvind
[Show all 7 contributors for this article]
(2021).
Structure prediction of honey bee vitellogenin: a multi-domain protein important for insect immunity.
FEBS Open Bio.
12(1),
p. 51–70.
doi:
10.1002/2211-5463.13316.
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Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg-laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full-length Vg (flVg) protein will provide insights regarding the structure–function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative-stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+-ion-binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid-body fitting to predict the relative position of high-resolution domains in a flVg model. This mapping represents the first experimentally validated full-length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.
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Streitlein, Luca; Canals, Mateu Montserrat & Krengel, Ute
(2024).
Structural analysis of chorismate mutase variants and interaction with partner enzymes.
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Sørensen, Henrik V.; Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare & Krengel, Ute
(2024).
New structural insights into bacterial colonization by Vibrio cholerae.
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Canals, Mateu Montserrat; Sørensen, Henrik Vinther; Oksanen, Esko & Krengel, Ute
(2022).
Neutron studies of the bacterial colonization factor GbpA.
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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.
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Canals, Mateu Montserrat; Sørensen, Henrik Vinther; Oksanen, Esko O. & Krengel, Ute
(2022).
Structural studies on GbpA using neutron scattering methods.
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Published
Mar. 19, 2025 9:41 AM
- Last modified
Mar. 19, 2025 9:42 AM
