STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF AUXIN INTERACTIONS WITH CANAVALIA LECTINS

Lectins from the Canavalia genus are highly conserved proteins whose hydrophobic regions may interact with non-carbohydrate ligands, including phytohormones. Here, we combined structural, computational, biophysical and physiological approaches to investigate the interaction of Canavalia brasiliensis lectin (ConBr) and Canavalia maritima lectin (ConM) with natural and synthetic auxins. Crystal structures of ConBr complexed with indole-3-acetic acid (IAA) and ConM complexed with indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were solved at high resolution, revealing a conserved auxin-binding site among Canavalia lectins. Although all auxins occupied the same binding pocket, differences in ligand orientation, hydrogen-bonding patterns and hydrophobic interactions resulted in distinct interaction energies. MFCC calculations identified the major residues contributing to ligand stabilization, while thermodynamic assays confirmed differences in binding affinity among the complexes. Structural comparisons revealed that natural and synthetic auxins occupy the same conserved binding site but assume different conformational and interaction states, with distinct ligand orientations and residue contacts, indicating that lectin–auxin recognition is a dynamic process involving multiple binding configurations. To investigate the biological significance of these interactions, Nicotiana tabacum leaf segments were infiltrated with IAA in the presence of increasing concentrations of ConM and cultivated in vitro. ConM promoted concentration-dependent physiological responses, with low concentrations stimulating callus formation and soluble protein accumulation, whereas higher concentrations reduced callus growth, increased lipid peroxidation and antioxidant enzyme activities, and altered the expression of auxin-responsive genes (ARF1, IAA14, IAA17 and SAUR59). The modulation of IAA14 expression was particularly dependent on lectin concentration, indicating a regulatory effect on auxin responses. Altogether, our results demonstrate that Canavalia lectins possess a conserved auxin-binding site with distinct affinities that are reflected in physiological responses, supporting the hypothesis that these proteins regulate auxin bioavailability and represent previously unrecognized modulators of plant development.

This work was supported by the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP-UNI-0210-00557.01.00/23), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-88881.165874/2018-01) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-305704/2025-7).