Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP Carboxyl Terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14- 3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements together with fluorescence quenching and mass spectrometry to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labelled WW3, WW4 and, to a lesser extent, WW2 domains and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo-form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.
This study was supported by the Czech Science Foundation (Projects 20-00058S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and by Grant Agency of Charles University (Project No.348421).