# Neutron crystallography study of host-pathogen recognition enhanced by hydrogen/deuterium exchange on carbohydrates

Theodore Arnaud, Chloe Tatol, Juliette M Devos, Lukas Gajdos, Sebastien

Vidal, Marek Korsák, Annabelle Varrot, Michaela Wimmerová, Sophie

Feuillastre, Matthew P Blakeley, et al.

► To cite this version:

Theodore Arnaud, Chloe Tatol, Juliette M Devos, Lukas Gajdos, Sebastien Vidal, et al. Neutron crystallography study of host-pathogen recognition enhanced by hydrogen/deuterium exchange on carbohydrates. 2025. hal-05345239

HAL Id: hal-05345239

https://hal.science/hal-05345239v1

Preprint submitted on 3 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Neutron Crystallography Study of Host-Pathogen Recognition Enhanced by Hydrogen/Deuterium Exchange on Carbohydrates

Theodore Arnaud¹,²,§, Chloé Tatol³,§, Juliette M. Devos⁴, Lukas Gajdos², Sébastien Vidal⁵, Marek Korsak⁶,⁷, Annabelle Varrot¹, Michaela Wimmerova⁶,⁷, Sophie Feuillastre³,*, Matthew P. Blakeley²,*, Anne Imberty¹,*

¹ Université Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

²Large Scale Structures Group, Institut Laue-Langevin, 71 Avenue des Martyrs, 38000 Grenoble, France

³ Université Paris-Saclay, CEA, Service de Chimie Bio-organique et Marquage, DMTS, 91191, Gif-sur-Yvette, France.

⁴Biology, Deuteration, Chemistry and Soft Matter Group, Institut Laue-Langevin, 71 Avenue des Martyrs, 38000 Grenoble, France.

⁵Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Université Paris-Saclay, Gif-sur-Yvette, 91198, France

⁶Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

⁷National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.

* Authors for correspondence: sophie.feuillastre@cea.fr; blakeleym@ill.fr; anne.imberty@cermav.cnrs.fr

§ These authors participated equally to the research
**Abstract**

Neutron macromolecular crystallography (NMX) is a unique tool for location of hydrogen atoms. Its application is greatly enhanced by the use of deuterium-labeled molecules. Human glycans are the targets of virulence factors from pathogens, such as the soluble lectins from the opportunistic bacterium *Pseudomonas aeruginosa*. Deuterated galactose was obtained by hydrogen isotope exchange and co-crystallized with a fully deuterated bacterial receptor, LecA, a lectin involved in *P. aeruginosa* tissue adhesion and biofilm formation. The structure of the complex determined using neutron diffraction reveals the positions of all hydrogen atoms, as deuterium, highlighting the important role of a charged histidine in the binding site, the bridging by a buried water molecule and the influence of the coordinating calcium ion on the adjacent hydrogen bonds. LecA is a target for pathoblockers and these structural details can assist in the design of glycomimetics for fighting multi-drug resistant infections.

**Keywords:** Neutron Macromolecular crystallography, Host-pathogen interaction, Carbohydrate, Hydrogen Isotope Exchange, Lectin
Introduction

Hydrogen (¹H) atoms are the most abundant constituents of biomolecules, accounting for almost
half of the atoms in protein molecules. They are essential for stabilizing biomolecular structures
and play crucial roles in the complex energy landscape of proteins.¹ Hydrogen atoms mediate
intra- and inter-molecular interactions through hydrogen bonds, CH-π or van der Waals
interactions. Deuterated biomolecules, in which some, or all hydrogen atoms are replaced by
deuterium (²H), are widely used in various domains, notably in drug development,²⁻³ mechanistic
studies of (bio)chemical processes⁴ and smart materials.⁵⁻⁶ Deuterated proteins are of particular
interest in structural biology, particularly for investigations using NMR,⁷ neutron diffraction,⁸ or
neutron scattering techniques.⁹

Deuterium-labeled small molecules, including carbohydrates, also have wide applications. For instance, labeled carbohydrate analogues are essential tools for the study of biomolecular dynamics (in vivo kinetics),¹⁰⁻¹¹ structural elucidation of biomacromolecules,¹² study of metabolism¹³⁻¹⁴ and are valuable chiral building blocks.¹⁵ Deuterated carbohydrates have also been used as detergents for studying membrane proteins in small-angle neutron scattering experiments.¹⁶ Since protein-carbohydrate interactions are involved in a wide range of biological processes, neutron crystallography studies of perdeuterated protein-carbohydrate complexes have been performed to determine the position of hydrogen atoms (as deuteriums) in carbohydrate binding sites. Perdeuterated D-xylulose and D-glucose were used for characterizing the enzyme mechanism in xylose isomerase¹⁷⁻¹⁸ and more recently, perdeuterated L-fucose was used to reveal the hydrogen bond networks in the binding sites of certain bacterial lectins.¹⁹⁻²⁰

While partial deuteration can be obtained via exchange of labile hydrogen atoms of O-H, N-H and S-H bonds by D₂O soaking or vapor diffusion, perdeuteration, i.e., the overall replacement of hydrogen atoms by deuterium atoms, requires stringent methodology. Biotechnology approaches are efficient for the production of recombinant deuterated proteins in bacteria after appropriate accommodation.²¹ For other biomolecules that are not directly produced through gene transcription, more complex synthetic strategies are required. In the case of carbohydrates, perdeuterated glucose and mannose were first obtained from algae grown in 99.6% D₂O medium,²²⁻²³ while perdeuterated cellulose and heparosan were produced from bacterial culture.²⁴⁻²⁵ Recently, we glyco-engineered *Escherichia coli* with the introduction of genes coding for GDP-fucose
synthesis enzymes, fucosyltransferase and fucosidase, for the production of perdeuterated L-fucose in a bioreactor.²⁶

Another complementary labeling method is hydrogen isotope exchange (HIE), which has revolutionized the field of labeled compounds, allowing late-stage labeling of a wide-range of organic molecules including drugs and complex biomolecules.²⁷⁻²⁸ However, only a few metal-mediated HIE methods have been implemented and optimized for the labeling of carbohydrates using either Raney Ni in D₂O²⁹⁻³⁰ or a non-commercial source of Ru/C (Scheme 1).³¹⁻³³ When using Raney Ni and D₂O on non-reducing carbohydrates, moderate deuterium atom incorporation was observed at the C2, C3 and C4 positions carrying free hydroxyl groups, while no labeling was observed at carbon atoms C1, C5. Moreover, the labeling of the C6 position was highly dependent of the activation mode used (see Scheme 1). Nevertheless, this technique has been employed for the deuteration of trehalose, as well as methyl pyranosides derived from D-glucose, D-galactose, and D-mannose.³⁴ Similarly, when using a non-commercial source of Ru/C in D₂O and under an atmosphere of H₂ gas, regio- and stereoselective H-to-D exchanges were observed on various pyranosides with good to quantitative deuterium incorporation on carbon atoms bearing hydroxyl groups. Despite their efficiency in delivering deuterated carbohydrates, both HIE methods suffer from drawbacks and limitations. These include the use of harsh experimental conditions in terms of activation or temperature, necessitating a strict control to prevent degradation or epimerization, or the use of a hazardous catalyst (Raney Ni) or of a non-commercially available one (Ru/C from N.E. Chemcat Corporation).

<figure><img src="image_2.png" /><figcaption>Scheme 1. State-of-the-art work using metal-mediated HIE for the labeling of carbohydrates</figcaption></figure>

Despite the challenges associated with the different strategies required to label biomolecules, the structural insights provided by deuterated molecules are worth the efforts. Neutron diffraction can precisely locate deuterium atoms and deuterated monosaccharides can be used for mapping these
atoms in the binding sites of lectins.³⁶ These glycan-binding proteins play key roles in self-
recognition and infection.³⁷ Their interaction with glycans is based on a combination of hydrogen-
bond networks and hydrophobic contacts, all mediated by hydrogen atoms³⁸, but structural data
obtained through neutron diffraction studies are still very limited.³⁹ Many pathogenic bacteria use
lectins to bind specifically to glycans present on human tissues⁴⁰ and our previous neutron structure
of LecB lectin from the opportunistic pathogen *Pseudomonas aeruginosa* in complex with L-fucose
demonstrated the structural basis of an unusually strong affinity for a monosaccharide.¹⁹ *P.
aeruginosa* produces another soluble lectin, LecA, that is specific for galactose and has been
identified as a virulence factor.⁴¹⁻⁴² The lectin binds specifically to α-galactoside exposed on
glycolipids in lungs or guts, resulting in cell and tissue invasion.⁴³⁻⁴⁴ As such, high-affinity
galactose-derived glycomimetics have been designed as competitors with anti-bacterial activity,
which is of strong interest in the view of multiresistant infection.⁴⁵⁻⁴⁷ The X-ray crystal structure
of LecA in complex with galactose demonstrated the bridging role of a calcium ion with the 3,4-
cis-hydroxyl groups of galactose,⁴⁸ and novel non-carbohydrate inhibitors targeting the calcium
ions are now being investigated.⁴⁹

The study presented here focuses on obtaining deuterated galactose for use in neutron
macromolecular crystallography (NMX) studies of LecA, in order to determine the role of
hydrogen atoms in the specificity of the protein towards the monosaccharide. Knowledge of these
crucial details can ultimately help towards the design of high-affinity inhibitors. Neutron structure
of the perdeuterated LecA/Gal complex provides unique information about protonation of amino
acids, such as an important histidine residue in the binding site, and reveals details of hydrogen
bonds close to the calcium ion as well as the role of water molecules.
## Results

### Hydrogen isotope exchange and characterization of resulting Gal-d<sub>5</sub> and Gal-d<sub>10</sub>

Methyl α-D-galactopyranoside-2,3,4,6,6'-d<sub>5</sub> (designated as α-OMe-Gal-d<sub>5</sub>) has been obtained by optimizing the protocol described by Sawama *et al.*,³³ notably by switching their specific source to a commercially available Ru/C source. Methyl α-D-galactopyranoside and the catalyst were mixed in D<sub>2</sub>O and placed under an atmosphere of D<sub>2</sub> gas at 90°C for 24 h, leading to full deuteration of all C-H bonds vicinal to free hydroxyl groups (namely the C2, C3, C4 and C6 positions). D-Galactose-2,3,4,6,6'-d<sub>5</sub> (designated as Gal-d<sub>5</sub>) has been obtained thereafter by hydrolysis of the methyl acetal. Soaking of Gal-d<sub>5</sub> in D<sub>2</sub>O gave rise to H/D exchange of all O-H bonds to O-D groups, resulting in the Gal-d<sub>10</sub> compound (Fig.1). All compounds have been analyzed using both NMR spectroscopy (¹H and ²H) and high-resolution mass spectrometry in order to determine isotopic enrichments at each position, as well as the total deuterium atom incorporation (Supp. Info).

<figure><img src="image_3.png" /><figcaption>Figure 1. Synthesis of labeled Gal-d<sub>5</sub> and Gal-d<sub>10</sub> through hydrogen isotope exchange. Isotopic enrichments are indicated between brackets at each labeled position.</figcaption></figure>

### Neutron structure of the D-LecA/Gal-d<sub>10</sub> complex

Recombinant perdeuterated LecA (D-LecA) was produced in *Escherichia coli* high cell-density cultures following adaptation to fully deuterated growth conditions,²¹ and was purified following established procedures (Supp Info).⁵⁰ Co-crystals of D-LecA/Gal-d<sub>10</sub> were obtained in sitting-drop trays using vapor-diffusion combined with regular crystal feeding to produce crystals of sufficient size for NMX experiments.⁵¹ A large co-crystal with volume of ~0.7 mm³ (Supp. Info) was used for room-temperature data collection, yielding neutron diffraction to 1.89 Å resolution and X-ray diffraction to 1.45 Å resolution (Table 1). The resulting structure after joint X-ray/neutron
refinement displays two tetramers in the asymmetric unit in space group *P*2₁ (i.e., eight chains, labeled A-H) (Supp. Info.). The eight chains have very similar structures (Cα RMSD < 0.23 Å) and the overall tetrameric biological assembly is also similar to previously published LecA crystal structures (e.g. PDB:1OKO, Cα RMSD < 0.23 Å) (Fig. 2A).

<table><caption>Table 1. Neutron and X-ray data collection and co-refinement statistics for the D-LecA/Gal-d₁₀ complex</caption><thead><tr><th></th><th>Neutrons</th><th>X-rays</th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td><td></td></tr><tr><td>Temperature</td><td>291K</td><td></td></tr><tr><td>Crystal size (mm³)</td><td>0.74</td><td></td></tr><tr><td>Space group</td><td><i>P</i>2<sub>1</sub></td><td></td></tr><tr><td><i>a</i>, <i>b</i>, <i>c</i> (Å)</td><td>50.80, 106.52, 83.30</td><td></td></tr><tr><td>α, β, γ (°)</td><td>90, 94.63, 90</td><td></td></tr><tr><td>Beamline, source</td><td>LADI-DALI, ILL</td><td>BM07-FIP2, ESRF</td></tr><tr><td>Wavelength (Å)</td><td>2.8–3.8</td><td>0.9202</td></tr><tr><td>Detector</td><td>Image plate</td><td>Pilatus 6M (DECTRIS)</td></tr><tr><td>Resolution (Å)</td><td>45.9 - 1.9 (2.0 - 1.9)</td><td>44.83-1.49 (1.52 - 1.49)</td></tr><tr><td>No of reflections</td><td></td><td></td></tr><tr><td>No. of unique reflections</td><td>50313 (6352)</td><td>142031 (7042)</td></tr><tr><td>R<sub>merge</sub> (I) (%)</td><td>20.6 (42.3)</td><td>9.0 (168.8)</td></tr><tr><td>R<sub>pim</sub> (I) (%)</td><td>12.5 (20.9)</td><td>6.5 (122.7)</td></tr><tr><td>Mean I/σ(I)</td><td>6.1 (2.4)</td><td>13.0 (0.9)</td></tr><tr><td>Completeness (%)</td><td>72.1 (62.6)</td><td>98.8 (99.4)</td></tr><tr><td>Multiplicity</td><td>3.1 (3.5)</td><td>5.4 (5.3)</td></tr><tr><td>CC1/2 (%)</td><td></td><td>99.8 (72.5)</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td></tr><tr><td>Resolution range (Å)</td><td>45.73 - 1.89</td><td>44.83 - 1.49</td></tr><tr><td>Reflections (used)</td><td>50298</td><td>141805</td></tr><tr><td>Reflections (test)</td><td>815</td><td>2739</td></tr><tr><td>R<sub>work</sub> (%)</td><td>21.5</td><td>15.7</td></tr><tr><td>R<sub>free</sub> (%)</td><td>27.8</td><td>18.5</td></tr><tr><td>RMSD in bond lengths (Å)</td><td></td><td>0.014</td></tr><tr><td>RMSD in bond angles (°)</td><td></td><td>1.7</td></tr><tr><td>No. of atoms / Average B factor (Å²)</td><td></td><td></td></tr><tr><td>Protein</td><td>14174 / 30.6</td><td></td></tr><tr><td>Ligand</td><td>200 / 31.7</td><td></td></tr><tr><td>Waters</td><td>848 /</td><td></td></tr><tr><td>Ramachandran statistics</td><td></td><td></td></tr><tr><td>Favored / Allowed / Outliers (%)</td><td>96.94 / 93.06 / 0</td><td></td></tr><tr><td>All-atom clash score</td><td>5.23</td><td></td></tr><tr><td>PDB code</td><td>9SQT</td><td></td></tr></tbody></table>

Values for the outer resolution shell are given in parentheses.
Crystallographic analysis of the deuteration of galactose

In all eight chains of the asymmetric unit, clear electron density and neutron scattering-length
density (hereafter referred to as neutron density) are observed for the galactose ligand (the average
2mF₀-DFc peak for galactose was above 2.8σ and 1.7σ in the X-ray electron density and neutron
density map, respectively). The galactose is in the expected ⁴C₁ chair conformation and presents
mostly in the α anomer configuration, which was therefore maintained during the structural
refinement procedure. Analysis of the neutron density maps confirmed deuteration on all oxygen
atoms and on positions C2, C3, C4 and C6. As expected, the neutron 2mF₀-DFc maps show no
peaks indicative of deuterium atoms at carbons C1 and C5, suggesting the presence of hydrogen
atoms at these two positions. This is in agreement with the results obtained by NMR and mass
spectrometry for the product from HIE synthesis (Fig. 2B). Neutron density is observed for
deuterium atoms on all hydroxyl groups of Gal-d₁₀ allowing for precise visualization of hydrogen
bonds (Fig. 2B and Fig. 3). Only the deuterium on O1 is not clearly localized, likely due to
cancellation of the nuclear scattering signal with the hydrogen atom of C1, and perhaps also the
local conformational flexibility at this position, as seen when superimposing the galactose residues
of the eight chains (Supp. Info).
<figure><img src="image_4.png" /><figcaption>Figure 2. Joint X-ray/neutron crystal structure of D-LecA/Gal-d₁₀ complex. (A) Simplified representation of one tetramer (chains A-D) with calcium ions represented as magenta spheres and galactose molecules as sticks (B) Neutron scattering-length density map shown as grey mesh (2mF₀-DFc contoured at 1.0σ) around the galactose ligand with deuterium atoms colored yellow and hydrogen atoms (at C1 and C5) colored white. (C) Neutron scattering-length density map in grey (2mF₀-DFc contoured at 1.0σ) and X-ray electron density map in blue (2mF₀-DFc contoured at 1.4σ) for charged histidine residues, His50 and His58, of chain A.</figcaption></figure>

Protonation states of amino acids

Inspection of the neutron density maps allows for a detailed analysis of the protonation states of
charged amino acids. The imidazole side chain of histidine has a pKₐ close to 6 and can exhibit
different charged states at physiological pH depending on the local environment. The LecA primary
sequence of amino acids contains two histidine residues, His50 and His58. In the LecA/Gal
complex (PDB: 1OKO), the NE2 atom of His50 forms a hydrogen bond with O6 of galactose,
while the ND1 atom of His58 interacts with a side chain of Asp52, thereby stabilizing the surface
loop comprising residues 50-53 involved in the binding site. Analysis of the neutron 2mF₀-DFc
density map of the deuterated complex indicates that both histidine residues have their two
imidazole nitrogen atoms protonated resulting in a cationic imidazolium (Fig. 2C). This
observation is consistent with the expected protonation state at the pH of the crystallization solution
(pD 5). Further confirmation that both His residues were bi-protonated was carried out by
performing refinement tests with different protonation states on the two histidine residues of the
eight monomers and comparing 2mF₀-DFc and mF₀-DFc omit neutron density maps (Supp. Info).
One aspartic acid residue (Asp100) is involved in both the coordination sphere of calcium and in
hydrogen bonding to O4 of galactose. Analysis of the neutron density indicates that this Asp is not
protonated.

Analysis of the hydrogen bond network in the binding site

The binding of galactose in the LecA recognition site results from both the coordination of a calcium ion and an extensive hydrogen bond network (Fig. 3). Continuous density is observed for all hydrogen bonds (above 1σ) with clear localization of the deuterium atoms on the hydroxyl groups of the galactose, and on asparagine and histidine side chains. Distances and angles characterizing the hydrogen bonds in the binding site are listed in Table 2.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** The galactose-binding site in the neutron structure of the D-LecA/Gal-d₁₀ complex (chain H). The hydrogen bonds are shown as orange dashed lines. The 2mF₀-DFc neutron density map (grey mesh) is contoured at 0.8σ. (A) Upper view of the binding site with Q53 omitted for clarity. (B) Alternative view including Q53 and displaying the coordination of the calcium ion.</figcaption></figure>
<table><caption>**Table 2.** Distances and angles for hydrogen bonds involving galactose and a conserved water molecule (W1) with standard deviations calculated across the eight chains of the asymmetric unit.</caption><thead><tr><th>Donor</th><th>Acceptor</th><th>Distance (Å)</th><th>Angle (°)</th></tr></thead><tbody><tr><td>Gal-O2D</td><td>W2-O</td><td>1.86 ± 0.15</td><td>152 ± 15</td></tr><tr><td>Gal-O3D</td><td>W3-O</td><td>1.91 ± 0.25</td><td>137 ± 20</td></tr><tr><td>Gal-O4D</td><td>Asp100-OE1</td><td>1.81 ± 0.1</td><td>145 ± 15</td></tr><tr><td>Gal-O6D</td><td>Gln53-OE1</td><td>1.99 ± 0.08</td><td>138 ± 15</td></tr><tr><td>His50-NE2</td><td>Gal-O6</td><td>1.91 ± 0.11</td><td>146 ± 5</td></tr><tr><td>W1-D1</td><td>Gal-O6</td><td>2.00 ± 0.12</td><td>152 ± 12</td></tr><tr><td>W1-D2</td><td>Pro51-O</td><td>1.81 ± 0.06</td><td>156 ± 10</td></tr><tr><td>Gln53-ND</td><td>W1-O</td><td>1.96 ± 0.05</td><td>160 ± 7</td></tr></tbody></table>

Galactose presents an axial orientation for its O4 hydroxyl group and the hydrogen bond between Gal-O4 and Asp100 is of key importance for the specificity. Gal-O3 accepts a hydrogen bond from the Asn107 side chain and donates one to a water molecule (W2), while Gal-O2 is oriented towards the solvent with hydrogen bonding to water molecule W3. The environment around Gal-O6, is of special interest since this hydroxyl group is accepting a hydrogen bond from protonated His50 and donating one to the side chain oxygen of Gln53. This flexible hydroxymethyl group is therefore trapped in only one conformation, which is unusual for a pyranose hydroxymethyl group. Furthermore, Gal-O6 also accepts a hydrogen bond from a water molecule (W1) that bridges between the galactose and three surrounding amino acids. This water molecule is trapped in a pocket on the protein surface and is conserved in all crystal structures of LecA (Fig. 4A). The neutron structure allows the unambiguous determination of its orientation with the water hydrogen atoms directed towards Gal-O6 and the carbonyl group of Pro51 while the water oxygen accepts a hydrogen bond from the main chain nitrogen of Gln53 (Fig. 4B).
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /><figcaption>Figure 4. Conserved water molecule W1 in the neutron structure of the D-LecA/Gal-d₁₀ complex. (A) Protein surface representation of the binding pocket. (B) Hydrogen-bond network around W1 with the 2mF₀-DFc neutron density map (grey mesh) contoured at 0.8σ.</figcaption></figure>

## Influence of calcium ion on galactose binding

In the carbohydrate-binding site of LecA, the calcium ion could not be directly observed in the neutron scattering density due to its low neutron scattering cross-section but its position is well defined in the X-ray electron density map. Although calcium coordination in LecA has been previously described,⁴⁸ our analysis revealed novel insights into the interaction network between galactose and LecA. Specifically, we observed that the orientation of the deuterium atoms on hydroxyl groups Gal-O3 and Gal-O4 are directed away from the calcium ion. Gal-OD3 participates in a hydrogen bond with water molecule W3, while Gal-OD4 forms a hydrogen bond with the OD1 atom of Asp100 (Fig. 3).

Based on previous structures as well as on the distances between galactose oxygens and side chain atoms of Asp100 and Asn107, it was proposed that both Gal-O3 and Gal-O4 establish bifurcated hydrogen bonds with OD1 and ND2 of Asn107, and OD1 and OD2 of Asp100, respectively. The neutron structure indicates a repulsing effect of the calcium ion on the hydrogens, influencing the hydrogen bond network.
Influence of His50 protonation on affinity

The hydrogen bond observed between His50 and Gal-O6, associated with a conserved orientation of W1 and its hydrogen bond network, depends on the protonation of the NE2 nitrogen atom of His50. At pH higher than 6, His50 is expected to be predominantly neutral, with only one nitrogen atom protonated and the hydrogen bond to Gal-O6 would be either absent or weaker due to neutral charge. This is of importance since physiological pH at the site of infection may vary from 5 to 7.4 depending on the tissue, inflammation state or other aspects.⁵² To further investigate the functional role of His50 in the binding site, the interaction of LecA with galactose was analyzed by isothermal titration calorimetry (ITC) at pH values of 5.0 and 7.3. Clear differences were observed with an affinity three times higher at pH 5.0 than at pH 7.3 (Fig. 5). The enthalpy of interaction is much stronger at pH 5.0 than at pH 7.3 (-44.1 kJ/mol versus -34.2 kJ/mol), confirming that hydrogen bonds are more numerous or stronger at acidic pH, when His50 is protonated.

<figure><img src="image_8.png" /></figure>

Figure 5. Titration of galactose into LecA solution by ITC at two different pH. Top: data obtained from 20 automatic injections, 2 µL each, of Gal (3 mM) into the cell containing LecA (0.3 mM). Lower: plot of the total heat released as a function of total ligand concentration for the titration shown above. The solid line represents the best least-squares fit for the obtained data.
Discussion and Conclusion

A combination of isotope-labeling methods enabled the production of deuterated LecA protein and deuterated galactose. The HIE method developed here is straightforward, easy to implement, and provided labeled galactose with maximized isotopic enrichments without degradation or racemization.

Analysis of the neutron structure of D-LecA/Gal-d₁₀ complex allowed to identify three key features: (1) the specific orientation of Gal-O3 and Gal-O4 hydroxyl groups induced by the presence of the calcium ion, (2) the critical role of the bridging water molecule in mediating interactions at the Gal-O6 position, and (3) the protonation state and involvement of His50 in ligand binding.

The direct involvement of a calcium ion in protein-carbohydrate interfaces has been observed in eight different classes of lectins,³⁸ three of them being from pathogenic microorganisms, i.e. LecA (this study) and LecB⁵³ from *P. aeruginosa* and epithelial adhesin from *Candida glabrata*.⁵⁴ Interestingly, no other metal ion has been observed in such a position. The calcium ion coordination sphere generally involves two adjacent *cis* hydroxyl groups, playing a role in specificity. It can also be involved in pH-dependent affinity of some human C-type lectins.⁵⁵ In the neutron structure of D-LecB/Fuc-d₁₂ complex, the presence of two calcium ions induces the formation of a low-barrier hydrogen bond between the fucose and the protein, participating in the unusually high affinity of this interaction.¹⁹ In the present structure, analysis of the neutron density demonstrates that the hydrogen atoms on the two coordinating galactose hydroxyl groups are pushed away from the calcium ion, reinforcing the hydrogen bonds between Gal-O4 and Asp100 that are crucial for specificity. This structural knowledge is important for the repurposing of metal-binding active compounds that have been recently demonstrated to be efficient non-carbohydrate glycomimetics for inhibition of LecA.⁴⁹,⁵⁶

Similarly, the importance of a conserved water molecule has been confirmed by the present study. The precise hydrogen bond network involving Gal-O6 and the water molecule trapped in the deep pocket of the binding site was determined. This water molecule (W1) is present in all crystal structures of LecA complexed with galactosides and galactose-derivatives. It has also a crucial role for binding the first non-carbohydrate glycomimetic to be identified, i.e., a cyano-catechol that
establishes a contact with W1 through its nitrogen atom.⁴⁹ The importance of this water molecule was assessed through a molecular dynamics study that demonstrated its strong stability (occupancy > 90%).⁵⁷ Displacement and replacement of a structural water molecule by a ligand is an efficient strategy to increase the affinity of a ligand via favorable enthalpy of binding.⁵⁸ Replacing W1 in LecA through ligand design would appear to be a promising strategy in the quest for high-affinity ligands for LecA, provided that this position can be reached.

The neutron structure of D-LecA/Gal-d₁₀ complex was obtained from crystallization performed at acidic pH (pD of 5) and the protonation of the two histidine residues are confirmed by the neutron density map analysis. The thermodynamic contributions measured by ITC correlate the low pH with higher affinity, that is consistent with the occurrence and/or enforcement of the hydrogen bond between His50 and Gal-O6. A more favorable enthalpy of binding with a difference of almost 10 kJ/mol is measured between pH 5 and pH 7.3 is only partially counterbalanced by unfavorable entropy. Higher affinity at low pH may favor the infectious process since surface liquid in lungs is more acidic in patients with cystic fibrosis.⁵⁹⁻⁶⁰

## Acknowledgements

T.A. is supported by both the French National Research Agency in the framework of the France 2030 investment program ANR-22-EXES-0001 and by the Institut Laue Langevin (ILL, Grenoble, France) and C.T. by the French National Research Agency ANR-22-CE07-0017 (Glyco-CD). M.W. and M.K. were supported by the Czech Science Foundation (21-29622S). Glyco@Alps (ANR-15-IDEX-02) is also acknowledged. The authors wish to thank the ILL for provision of beamtime and technical support on the LADI-DALI beamline, for access to the Deuteration Laboratory platform (ILL D-Lab) and for studentship funding to M.K. We acknowledge the ESRF (Grenoble, France) for provision of synchrotron radiation beamtime on beamline BM07-FIP2, supported by the French ANR PIA3 (France 2030) EquipEx+ project MAGNIFIX under grant agreement ANR-21-ESRE-0011. We thank Sylvain Engilberge for support during the experiment. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by
FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes
graduate school CBH-EUR-GS (ANR-17-EURE-0003).

**Supporting information**

Material and methods and experimental details for synthesis and characterization, as well as
supplemental figures of the crystal structures.

References

1. Engler, N.; Ostermann, A.; Niimura, N.; Parak, F. G., Hydrogen atoms in proteins: positions and dynamics. *Proc. Natl. Acad. Sci. U. S. A.* **2003**, *100*, 10243-8.

2. Atzrodt, J.; Derdau, V.; Kerr, W. J.; Reid, M., Deuterium- and Tritium-Labelled Compounds: Applications in the Life Sciences. *Angew. Chem. Int. Ed. Engl.* **2018**, *57*, 1758-1784.

3. Kopf, S.; Bourriquen, F.; Li, W.; Neumann, H.; Junge, K.; Beller, M., Recent Developments for the Deuterium and Tritium Labeling of Organic Molecules. *Chem. Rev.* **2022**, *122*, 6634-6718.

4. Fitzpatrick, P. F., Combining solvent isotope effects with substrate isotope effects in mechanistic studies of alcohol and amine oxidation by enzymes. *Biochim. Biophys. Acta* **2015**, *1854*, 1746-55.

5. Grimm, J. B.; Xie, L.; Casler, J. C.; Patel, R.; Tkachuk, A. N.; Falco, N.; Choi, H.; Lippincott-Schwartz, J.; Brown, T. A.; Glick, B. S.; Liu, Z.; Lavis, L. D., A General Method to Improve Fluorophores Using Deuterated Auxochromes. *JACS Au* **2021**, *1*, 690-696.

6. Shao, M.; Keum, J.; Chen, J.; He, Y.; Chen, W.; Browning, J. F.; Jakowski, J.; Sumpter, B. G.; Ivanov, I. N.; Ma, Y. Z.; Rouleau, C. M.; Smith, S. C.; Geohegan, D. B.; Hong, K.; Xiao, K., The isotopic effects of deuteration on optoelectronic properties of conducting polymers. *Nat Commun* **2014**, *5*, 3180.

7. Tugarinov, V., Indirect use of deuterium in solution NMR studies of protein structure and hydrogen bonding. *Prog. Nucl. Magn. Reson. Spectrosc.* **2014**, *77*, 49-68.

8. Blakeley, M. P.; Podjarny, A. D., Neutron macromolecular crystallography. *Emerg. Top. Life Sci.* **2018**, *2*, 39-55.

9. Mahieu, E.; Gabel, F., Biological small-angle neutron scattering: recent results and development. *Acta Crystallogr D Struct Biol* **2018**, *74*, 715-726.
10. Bier, D. M.; Arnold, K. J.; Sherman, W. R.; Holland, W. H.; Holmes, W. F.; Kipnis, D. M., *In-vivo* measurement of glucose and alanine metabolism with stable isotopic tracers. *Diabetes* **1977**, *26*, 1005-15.

11. Indurugalla, D.; Bennet, A. J., A kinetic isotope effect study on the hydrolysis reactions of methyl xylopyranosides and methyl 5-thioxylopyranosides: oxygen versus sulfur stabilization of carbenium ions. *J. Am. Chem. Soc.* **2001**, *123*, 10889-98.

12. Vallurupalli, P.; Scott, L.; Hennig, M.; Williamson, J. R.; Kay, L. E., New RNA labeling methods offer dramatic sensitivity enhancements in 2H NMR relaxation spectra. *J. Am. Chem. Soc.* **2006**, *128*, 9346-7.

13. De Feyter, H. M.; de Graaf, R. A., Deuterium metabolic imaging - Back to the future. *J. Magn. Reson.* **2021**, *326*, 106932.

14. Lowery, R.; Gibson, M. I.; Thompson, R. L.; Fullam, E., Deuterated carbohydrate probes as 'label-free' substrates for probing nutrient uptake in mycobacteria by nuclear reaction analysis. *Chem Commun (Camb)* **2015**, *51*, 4838-41.

15. Milijkovic, M., *Carbohydrates: Synthesis, Mechanisms, and Stereoelectronic Effects*. Springer: New York, 2009.

16. Midtgaard, S. R.; Darwish, T. A.; Pedersen, M. C.; Huda, P.; Larsen, A. H.; Jensen, G. V.; Kynde, S. A. R.; Skar-Gislinge, N.; Nielsen, A. J. Z.; Olesen, C.; Blaise, M.; Dorosz, J. J.; Thorsen, T. S.; Venskutonyte, R.; Krintel, C.; Moller, J. V.; Frielinghaus, H.; Gilbert, E. P.; Martel, A.; Kastrup, J. S.; Jensen, P. E.; Nissen, P.; Arleth, L., Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering. *FEBS J* **2018**, *285*, 357-371.

17. Kovalevsky, A. Y.; Hanson, L.; Fisher, S. Z.; Mustyakimov, M.; Mason, S. A.; Forsyth, V. T.; Blakeley, M. P.; Keen, D. A.; Wagner, T.; Carrell, H. L.; Katz, A. K.; Glusker, J. P.; Langan, P., Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study. *Structure* **2010**, *18*, 688-99.

18. Kovalevsky, A. Y.; Katz, A. K.; Carrell, H. L.; Hanson, L.; Mustyakimov, M.; Fisher, S. Z.; Coates, L.; Schoenborn, B. P.; Bunick, G. J.; Glusker, J. P.; Langan, P., Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose. *Biochemistry* **2008**, *47*, 7595-7.

19. Gajdos, L.; Blakeley, M. P.; Haertlein, M.; Forsyth, V. T.; Devos, J. M.; Imberty, A., Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding. *Nat. Commun.* **2022**, *13*, 194.

20. Gajdos, L.; Blakeley, M. P.; Kumar, A.; Wimmerová, M.; Haertlein, M.; Forsyth, V. T.; Imberty, A.; Devos, J. M., Visualisation of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions. *Structure* **2021**, *29*, 1003-1013.
21. Haertlein, M.; Moulin, M.; Devos, J. M.; Laux, V.; Dunne, O.; Forsyth, V. T., Biomolecular Deuteration for Neutron Structural Biology and Dynamics. *Methods Enzymol.* **2016**, *566*, 113-57.

22. Chorney, W.; Scully, N. J.; Crespi, H. L.; Katz, J. J., The growth of algae in deuterium oxide. *Biochim. Biophys. Acta* **1960**, *37*, 280-7.

23. Gatley, S. J.; Wess, M. M.; Govoni, P. L.; Wagner, A.; Katz, J. J.; Friedman, A. M., Deuterioglucose: alteration of biodistribution by an isotope effect. *J. Nucl. Med.* **1986**, *27*, 388-94.

24. O'Neill, H.; Shah, R.; Evans, B. R.; He, J.; Pingali, S. V.; Chundawat, S. P.; Jones, A. D.; Langan, P.; Davison, B. H.; Urban, V., Production of bacterial cellulose with controlled deuterium-hydrogen substitution for neutron scattering studies. *Methods Enzymol.* **2015**, *565*, 123-46.

25. Cress, B. F.; Bhaskar, U.; Vaidyanathan, D.; Williams, A.; Cai, C.; Liu, X.; Fu, L.; V, M. C.; Zhang, F.; Mousa, S. A.; Dordick, J. S.; Koffas, M. A. G.; Linhardt, R. J., Heavy Heparin: A Stable Isotope-Enriched, Chemoenzymatically-Synthesized, Poly-Component Drug. *Angew. Chem. Int. Ed. Engl.* **2019**, *58*, 5962-5966.

26. Gajdos, L.; Forsyth, V. T.; Blakeley, M. P.; Haertlein, M.; Imberty, A.; Samain, E.; Devos, J. M., Production of perdeuterated fucose from glyco-engineered bacteria. *Glycobiology* **2021**, *31*, 151-158

27. Mullard, A., FDA approves first deuterated drug. *Nat Rev Drug Discov* **2017**, *16*, 305.

28. Schmidt, C., First deuterated drug approved. *Nat. Biotechnol.* **2017**, *35*, 493-494.

29. Bokatzian-Johnson, S. S.; Maier, M. L.; Bell, R. H.; Alston, K. E.; Le, B. Y.; Cioffi, E. A., Facile C-H bond activation for deuterium and tritium labeling of glycoconjugates conducted in ultrasonic and microwave fields: a review. *J. Label. Compd. Radiopharm.* **2007**, *50*, 380-383.

30. Heys, J. R., Nickel-catalyzed hydrogen isotope exchange. *J. Label Compd. Radiopharm.* **2010**, *53*, 716-721.

31. Fujiwara, Y.; Iwata, H.; Sawama, Y.; Monguchi, Y.; Sajiki, H., Method for regio-, chemo- and stereoselective deuterium labeling of sugars based on ruthenium-catalyzed C-H bond activation. *Chem Commun (Camb)* **2010**, *46*, 4977-9.

32. Sakurada, N.; Sasaki, D.; Ono, M.; Yamada, T.; Ikawa, T.; Sajiki, H. D., Development of Site- and Stereoselective Continuous Flow Deuterium Labelling Method for Carbohydrates Using High Dispersion Effect towards Ru/C of Hydrogen Flow. *React. Chem. Eng.* **2025**, *10*, 777-781.

33. Sawama, Y.; Yabe, Y.; Iwata, H.; Fujiwara, Y.; Monguchi, Y.; Sajiki, H., Stereo- and regioselective direct multi-deuterium-labeling methods for sugars. *Chemistry* **2012**, *18*, 16436-42.
34. Koch, H. J.; Stuart, R. S., The catalytic C-deuteration of some carbohydrate derivatives. *Carbohydr. Res.* **1978**, *67*, 341-348.

35. Koch, H. J. S., R.S., The synthesis of per-C-deuterated D-glucose. *Carbohydr. Res.* **1978**, *64*, 127-134.

36. Helliwell, J. R., Relating protein crystal structure to ligand-binding thermodynamics. *Acta Cryst.* **2022**, *F78*, 403-407.

37. Lis, H.; Sharon, N., Lectins: Carbohydrate-specific proteins that mediate cellular recognition. *Chem. Rev.* **1998**, *98*, 637-674.

38. Angulo, J.; Zimmer, J.; Imberty, A.; Prestergard, J., Structural biology of glycan recognition. In *Essentials of Glycobiology, Fourth Edition*, Varki, A.; Prestegard, J. H.; Schnaar, R. L.; Seeberger, P. H.; Cummings, R. D.; Esko, J. D.; Stanley, P.; Hart, G. W.; Aebi, M.; Mohnen, D.; Kinoshita, T.; Packer, N. H., Eds. *Cold Spring Harbor Laboratory Press: Cold Spring Harbor* 2022; pp 403-418.

39. Lisacek, F.; Schnider, B.; Imberty, A., Tools for structural lectinomics: From structures to lectomes. *hal-04979945 2025*, *7*, 100154.

40. Farez, M.; Imberty, A.; Titz, A., Bacterial lectins: multifunctional tools in pathogenesis and possible drug targets. *Trends Microb. in press*.

41. Bajolet-Laudinat, O.; Girod-de Bentzmann, S.; Tournier, J. M.; Madoulet, C.; Plotkowski, M. C.; Chippaux, C.; Puchelle, E., Cytotoxicity of *Pseudomonas aeruginosa* internal lectin PA-I to respiratory epithelial cells in primary culture. *Infect. Immun.* **1994**, *62*, 4481-4487.

42. Laughlin, R. S.; Musch, M. W.; Hollbrook, C. J.; Rocha, F. M.; Chang, E. B.; Alverdy, J. C., The key role of Pseudomonas aeruginosa PA-I lectin on experimental gut-derived sepsis. *Ann. Surg.* **2000**, *232*, 133-42.

43. Chemani, C.; Imberty, A.; de Bentzman, S.; Pierre, P.; Wimmerová, M.; Guery, B. P.; Faure, K., Role of LecA and LecB lectins in *Pseudomonas aeruginosa* induced lung injury and effect of carbohydrates ligands. *Infect. Immun.* **2009**, *77*, 2065-2075.

44. Eierhoff, T.; Bastian, B.; Thuenauer, R.; Madl, J.; Audfray, A.; Aigal, S.; Juillot, S.; Rydell, G. E.; Müller, S.; de Bentzmann, S.; Imberty, A.; Fleck, C.; Römer, W., A lipid zipper triggers bacterial invasion. *Proc. Natl. Acad. Sci. U. S. A.* **2014**, *111*, 12895-12900.

45. Cecioni, S.; Imberty, A.; Vidal, S., Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands. *Chem. Rev.* **2015**, *115*, 525-561.

46. Leusmann, S.; Menova, P.; Shanin, E.; Titz, A.; Rademacher, C., Glycomimetics for the inhibition and modulation of lectins. *Chem. Soc. Rev.* **2023**, *52*, 3663-3740.

47. Wojtczak, K.; Byrne, J. P., Structural Considerations for Building Synthetic Glycoconjugates as Inhibitors for Pseudomonas aeruginosa Lectins. *ChemMedChem* **2022**, *17*, e202200081.
48. Cioci, G.; Mitchell, E. P.; Gautier, C.; Wimmerova, M.; Sudakevitz, D.; Pérez, S.; Gilboa-Garber, N.; Imberty, A., Structural basis of calcium and galactose recognition by the lectin PA-IL of *Pseudomonas aeruginosa*. *FEBS Lett.* **2003**, *555*, 297-301.

49. Kuhaudomlarp, S.; Siebs, E.; Shanina, E.; Topin, J.; Joachim, I.; da Silva Figueiredo Celestino Gomes, P.; Varrot, A.; Rognan, D.; Rademacher, C.; Imberty, A.; Titz, A., Non-carbohydrate glycomimetics as inhibitors of calcium(II)-binding lectins. *Angew. Chem. Int. Ed.* **2021**, *60*, 2-13.

50. Blanchard, B.; Nurisso, A.; Hollville, E.; Tétaud, C.; Wiels, J.; Pokorná, M.; Wimmerová, M.; Varrot, A.; Imberty, A., Structural basis of the preferential binding for globo-series glycosphingolipids displayed by *Pseudomonas aeruginosa* lectin I (PA-IL). *J. Mol. Biol.* **2008**, *383*, 837-853.

51. Budayova-Spano, M.; Koruza, K.; Fisher, Z., Large crystal growth for neutron protein crystallography. *Methods Enzymol.* **2020**, *634*, 21-46.

52. Torres, I. M.; Demirdjian, S.; Vargas, J.; Goodale, B. C.; Berwin, B., Acidosis increases the susceptibility of respiratory epithelial cells to Pseudomonas aeruginosa-induced cytotoxicity. *Am J Physiol Lung Cell Mol Physiol* **2017**, *313*, L126-L137.

53. Mitchell, E.; Houles, C.; Sudakevitz, D.; Wimmerova, M.; Gautier, C.; Pérez, S.; Wu, A. M.; Gilboa-Garber, N.; Imberty, A., Structural basis for oligosaccharide-mediated adhesion of *Pseudomonas aeruginosa* in the lungs of cystic fibrosis patients. *Nature Struct. Biol.* **2002**, *9*, 918-921.

54. Ielasi, F. S.; Decanniere, K.; Willaert, R. G., The epithelial adhesin 1 (Epa1p) from the human-pathogenic yeast Candida glabrata: structural and functional study of the carbohydrate-binding domain. *Acta Crystallogr. D. Biol. Crystallogr.* **2012**, *68*, 210-7.

55. Joswig, J. O.; Anders, J.; Zhang, H.; Rademacher, C.; Keller, B. G., The molecular basis for the pH-dependent calcium affinity of the pattern recognition receptor langerin. *J. Biol. Chem.* **2021**, *296*, 100718.

56. Shanina, E.; Kuhaudomlarp, S.; Siebs, E.; Fuchsberger, F.; Denis, M.; da Silva Figueiredo Celestino Gomes, P.; Clausen, M. H.; Seeberger, P. H.; Rognan, D.; Titz, A.; Imberty, A.; Rademacher, C., Targeting undruggable carbohydrate recognition sites through focused fragment library design. *Commun. Chem.* **2022**, *5*, 64.

57. Nurisso, A.; Blanchard, B.; Audfray, A.; Rydner, L.; Oscarson, S.; Varrot, A.; Imberty, A., Role of water molecules in structure and energetics of *Pseudomonas aeruginosa* PA-IL lectin interacting with disaccharides. *J. Biol. Chem.* **2010**, *285*, 20316-20327.

58. Matricon, P.; Suresh, R. R.; Gao, Z. G.; Panel, N.; Jacobson, K. A.; Carlsson, J., Ligand design by targeting a binding site water. *Chem. Sci.* **2020**, *12*, 960-968.

59. Simonin, J.; Bille, E.; Crambert, G.; Noel, S.; Dreano, E.; Edwards, A.; Hatton, A.; Pranke, I.; Villeret, B.; Cottart, C. H.; Vrel, J. P.; Urbach, V.; Baatallah, N.; Hinzpeter, A.; Golec, A.;
Touqui, L.; Nassif, X.; Galietta, L. J. V.; Planelles, G.; Sallenave, J. M.; Edelman, A.; Sermet-Gaudelus, I., Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis. *Sci Rep* **2019**, *9*, 6516.

60. Tang, X. X.; Ostedgaard, L. S.; Hoegger, M. J.; Moninger, T. O.; Karp, P. H.; McMenimen, J. D.; Choudhury, B.; Varki, A.; Stoltz, D. A.; Welsh, M. J., Acidic pH increases airway surface liquid viscosity in cystic fibrosis. *J. Clin. Invest.* **2016**, *126*, 879-91.