Domain-specific variants reshape terpene synthase conformational dynamics and ligand binding in Coffea arabica populations from Saudi Arabia

Coffea arabica is an allotetraploid crop with limited genetic diversity, while terpene synthases (TPSs) contribute to specialized metabolism, ecological adaptation, and aroma formation. Whole-genome sequencing of Saudi Arabian C. arabica identified variants in five of 69 TPS genes, including eight missense substitutions in LOC113689980 (XP_071904867.1). Their structural consequences and the influence of TPS lid architecture were investigated using molecular modeling, docking, and three independent 300-ns molecular dynamics simulations per system. p.Glu424Val in 04867_cof_E424V_FPF preserved the global protein fold but altered ligand dynamics, Mg²⁺ coordination, and long-range catalytic coupling. The eight-substitution 04867_cof_mutant_FPF construct reduced ligand RMSD relative to 04867_cof_E424V_FPF (3.162 ± 0.743 vs. 3.831 ± 0.273 Å) and partially restored residue-424 coupling with DDXXD (− 0.138 to + 0.121; WT + 0.245) and NSE/DTE (+ 0.152 to + 0.208; WT + 0.404). However, the predefined statistical criterion for compensatory epistasis was not met (BH-FDR q = 0.549), indicating directional rather than statistically supported compensation. Lid architecture produced a stronger effect: the lid-containing 09630_cof_FPF complex showed significantly lower ligand RMSD (2.910 ± 0.122 vs. 4.166 ± 0.311 Å; q = 0.011) and ligand-contact residue RMSF (0.736 ± 0.064 vs. 1.973 ± 0.158 Å; q = 0.003) than the lid-absent 07178_cof_FPF. PCA/FEL further revealed system- and replicate-dependent redistribution of conformational ensembles rather than uniform changes in global flexibility. Collectively, these findings show that naturally occurring TPS variation can remodel catalytic-site dynamics while preserving overall structural integrity, whereas lid architecture strongly influences ligand confinement. The computationally constructed multi-variant model showed directional compensation in structural and dynamical properties relative to 04867_cof_E424V_FPF; however, this trend was neither statistically supported as positive epistasis nor reflected as stable energetic compensation in the ensemble QM/MM analysis. These findings provide a mechanistic basis for future haplotype-resolved analyses and experimental validation.