Neutron Crystallography Maps the Interaction Landscape of Pseudomonas aeruginosa LecA with Deuterated Aromatic β-Galactoside

Abstract

Aromatic derivatives of β-galactosides are potent inhibitors of the LecA lectin, a key virulence factor of Pseudomonas aeruginosa, involved in host recognition, tissue adhesion and biofilm formation. This opportunistic bacterial pathogen is considered a serious global health threat, particularly due to the emergence of multidrug-resistant strains. Anti-adhesive and anti-biofilm compounds represent promising alternatives or complements to conventional antibiotic therapies. The design of such pathoblockers requires detailed structural insights into their interactions with the target protein. Neutron macromolecular crystallography (NMX) provides complementary structural information to X-ray diffraction (XRD), by enabling the localization of hydrogen atoms, but typically requires the use of deuterium-labeled molecules. We developed a unique approach combining bacterial production of deuterated galactose and its functionalization with a deuterated phenyl group, resulting in the first synthesis of perdeuterated phenyl-β-galactoside, PheβGal-d16. The structure of the deuterated bacterial receptor LecA in complex with PheβGal-d16 was determined using neutron diffraction, providing experimental localization of hydrogen atoms involved in hydrophobic contacts with the Pro38 and Gln53 residues, and in the unusual T-shaped π-stacking interaction with His50. These observations rationalize the 10-fold affinity increase measured for an aromatic β-galactoside compared to galactose.