Structural characterization of human glutamine synthetase and its supramolecular organization

Human glutamine synthetase (hGS) is a central metabolic enzyme in nitrogen homeostasis, whose canonical form consists of a decamer formed by the association of two pentameric rings. Although its decameric organization is well established, the structural mechanisms underlying its assembly into higher-order supramolecular states and the conformational consequences of this transition remain poorly understood. Here, we investigated the supramolecular organization of hGS by combining solution-based biophysical characterization, high-resolution cryo-electron microscopy (cryo-EM), and conformational variability analyses. SEC-MALS and mass photometry revealed the coexistence of multiple oligomeric populations, indicating a dynamic equilibrium between decamers and higher-order species. We determined the cryo-EM structures of the hGS decamer and filament at global resolutions of 2.23 Å and 2.67 Å, respectively. The filament structure reveals a face-to-face association between consecutive decamers, mediated by a recurring interface primarily involving residues K52, C53, and E55. Beyond static structural characterization, three-dimensional variability analyses were employed to explore the reorganization of the conformational space accessible to the enzyme. Characterization of these conformational trajectories suggests that inter-subunit dynamics may contribute both to filament assembly and stabilization and to structural changes potentially related to the catalytic mechanism. Together, our results establish a structural and conformational framework for the supramolecular organization of hGS and suggest that filamentation emerges from the interplay between oligomeric equilibrium, specific protein interfaces, and conformational dynamics.