Structural Evidence for Occupancy-Dependent Inter-Site Coupling in Human Glutathione Synthetase

Abstract

Human glutathione synthetase (hGS) is a negatively cooperative ATP-grasp enzyme that catalyzes the final step in the biosynthesis of glutathione, a tripeptide antioxidant critical for life. hGS functions as an obligate homodimer with one active site per subunit; the two active sites are separated by [~]40 Angstroms. How ligand binding in one subunit reshapes the distant partner active site has remained a central unresolved question in understanding hGS regulation. This study provides the first atomistic model of ligand-dependent inter-subunit communication underlying negative cooperativity in hGS. Using atomistic simulations and dynamical network analysis, this study reveals how reactant- and product-bound states remodel the empty partner active site, redistribute inter-subunit interactions, and organize long-range communication between the two active sites. The product-bound/partner-empty state displayed a larger and less hydrated empty active site, demonstrating that ligand identity in one subunit alters both the geometry and solvent environment of the opposite site. Changes in ligand-dependent interactions are distributed across the dimer interface, with prominent contributions from the 42-46 interface region, the 11-30 region, and the 212-236 helical/interface region. Suboptimal path analysis shows product- and reactant-bound states share a communication scaffold, with 64.1% of transmission residues common to both pathways, 30.8% product-specific, and 5.1% reactant-specific. Together, the present results establish a detailed structural framework for hGS negative cooperativity in which ligand binding remodels a distributed allosteric network linking substrate-binding loops, the dimer interface, and the partner active site. More broadly, this work demonstrates how atomistic simulations can resolve long-range active-site coupling in multimeric enzymes and provides a foundation for experimental tests of allosteric transmission in hGS.