Calcium-mediated modulation of ultra-low-ester pectin-gluten interactions: subunit-level mechanisms revealed using HMW-GS deletion lines and molecular dynamics simulations.

Interactions between dietary fibers and gluten critically define the structural integrity and textural performance of wheat-based foods. This study elucidates how calcium ions (Ca2+) orchestrate the molecular interplay between low-ester pectin (LEP, 13.5% DE) and high-molecular-weight glutenin subunits (HMW-GSs) at the Glu-D1 locus (Dx2 and Dy12). Through an integrated approach combining multi-scale experimental analyses (SE-HPLC, RP-HPLC, FTIR, Raman spectroscopy, CLSM, and rheology) with all-atom molecular dynamics simulations, we demonstrate that LEP alone disrupts disulfide cross-linking, β-sheet order, and hydrophobic packing within gluten networks, while Ca2+ reverses these perturbations by forming "egg-box" ionic bridges between LEP carboxyl groups and acidic residues on HMW-GSs. The extent of this structural recovery was highly subunit-dependent: Dx2 displayed superior structural compactness, stronger Ca2+ coordination, and greater electrostatic stabilization than y-type Dy12, which remained more flexible and less responsive. Consequently, Dx2 functions as the primary structural anchor enabling Ca2+-mediated recovery of polymerization, hydrogen bonding, and viscoelastic elasticity. These findings reveal that Dx2 impart reversible conformational resilience under Ca2+-regulated pectin coupling, providing a mechanistic framework for optimizing LEP fortification and informing subunit-oriented wheat breeding strategies to achieve nutritionally enhanced yet structurally stable fiber-enriched products.