How Structure and Dynamic Shape Affinity in Human Galectin-3: An NMR Perspective

Galectins comprise a large family of β-galactoside-binding proteins, classified into three structural subtypes: prototypic, chimeric, and tandem-repeat. Galectin-3 (Gal-3) is the only known chimeric member, distinguished by an intrinsically disordered N-terminal domain responsible for oligomerization, and a C-terminal carbohydrate recognition domain (CRD). Depending on the cellular context, Gal 3 plays a dual role in tumor progression, exhibiting both pro-survival and anti tumor functions. This functional versatility has made Gal-3 a highly attractive, yet complex, target for therapeutic intervention. The molecular basis underlying Galectin-3 (Gal-3) ligand selectivity remains incompletely understood, despite clear evidence of its differential binding affinities for various carbohydrate structures. Current studies suggest that this selectivity arises from a complex interplay between protein conformational dynamics (Diehl et al., 2010) and solvent-mediated interactions (Modenutti et al., 2019); however, the precise structural determinants remain elusive. To address this gap, we employed solution-state NMR spectroscopy to investigate the binding mechanism of the carbohydrate recognition domain of human Gal-3 (CRD-Gal3), focusing on conformational dynamics associated with high- and low-affinity ligand interactions. Recombinant CRD-Gal3 was expressed in Escherichia coli BL21(DE3) using a pET28a vector and purified via a two-step protocol comprising nickel-affinity chromatography (HisTrap FF) followed by size exclusion chromatography (SEC), yielding a properly folded protein suitable for high-resolution structural analysis. Our NMR-based strategy integrated chemical shift perturbation (CSP) mapping to identify ligand-binding interfaces; backbone dynamics analysis via relaxation measurements (R₁, R₂, and {^1H}-^15N heteronuclear NOE); solvent accessibility profiling through CLEANEX-PM experiments; assessment of hydrogen bond networks via temperature coefficient analysis; and conformational landscape mapping using high-pressure NMR spectroscopy. The results reveal that ligand binding induces distinct changes in the chemical environment and alters the dynamic behavior of CRD-Gal3. Although no major global conformational rearrangements are observed between high- and low affinity complexes, localized differences—particularly in binding site flexibility and solvent reorganization—likely contribute to the observed variations in binding affinity. These findings offer novel mechanistic insights into the structural basis of Gal-3’s carbohydrate recognition specificity and establish a robust experimental framework for the future design of Gal-3 modulators with therapeutic potential.