Dynamic networks connect the USP14 active site region with the proteasome interaction surface.

Ubiquitin-specific protease 14 (USP14) is a member of the USP family responsible for the catalytic removal of ubiquitin (Ub) from proteins directed to the proteasome, implicated in the pathogenesis of neurodegeneration and cancer. Crystallography and cryo-EM analysis have identified loop regions crucial for the deubiquitinase activity of USP14, specifically those involved in Ub and proteasome binding. However, the structural changes in USP14 upon ligand binding to these regions are minimal, indicating significant yet uncharacterized dynamic contributions to its function. In this study, through structural and dynamical NMR experiments and functional evaluation, we demonstrate that small mutations designed to impact Ub binding and catalytic activity without disturbing the USP structure display both local and long-range effects. The affected residues connect the catalytic site and the Ub binding region with the proteasome interaction surface through a network of loops, which show varied dynamics on the ps-ms time scale. Collectively, our findings experimentally reveal different aspects of dynamic connections within USP14, suggesting the presence of allosteric networks that link enzyme activity with regulatory function. The identification of coupled clusters of possible allostery participants in the free USP domain provides new insights into the dynamic regulation of USP14, with potential implications for understanding its role in cellular processes.