Evaluation of ADP Glucose Pyrophosphorylase Subunit Interaction in Wheat by Binding Free Energy Calculations

ADP-glucose pyrophosphorylase (AGPase; E.C. 2.7.7.27) is the rate-limiting enzyme catalyzing the first committed step of starch biosynthesis in higher plants. The enzyme functions as a heterotetramer comprising two large (LS) and two small (SS) subunits that share 47.02% sequence identity in wheat. To elucidate the structural mechanism underlying heterotetramer assembly, we generated six possible dimeric conformations based on two-fold symmetry, three side-by-side (D1, D2, D3), and three upside-down (D4, D5, D6) orientations and evaluated their stability through all-atom molecular dynamics (MD) simulations combined with MM-GBSA and MM-PBSA free energy analyses. Among all configurations, the D2 heterodimer emerged as the most stable, exhibiting the lowest binding free energy (-15.2 kcal·mol⁻¹), largest interface area (1757.2 Ų), and strongest predicted affinity (Kd = 2.1 × 10⁻¹¹ M). Interaction energy analysis revealed that D2 stability is primarily governed by an extensive network of 25 hydrogen bonds and seven salt bridges at the LS-SS interface. Together, these results provide the first comprehensive molecular insight into the assembly and stabilization of wheat AGPase, a central determinant of starch biosynthetic efficiency. These results provide the first deep-learning-based molecular model of wheat AGPase, offering detailed structural insight into its subunit assembly and stability mechanisms. By identifying key interfacial residues that govern complex formation, this study establishes a foundation for rational protein engineering aimed at enhancing AGPase thermostability and catalytic efficiency, traits directly linked to improved starch accumulation and grain yield in cereal crops.