Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

R. Joshi1, 2, T. Obsil1, 2, V. Obsilova1

1 Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, 252 50 Vestec, Czech Republic.

2 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843, Prague, Czech Republic.


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.

1. J. Manning, S. Kumar, Trends Biochem. Sci. 43, (2018), 635647.

2. T. Ichimura, T. Isobe, J Biol Chem., 280, (2005), 13187-94.

3. P. Pohl, R Joshi, O. Petrvalska, T. Obsil, V. Obsilova. Commun Biol., 4, (2021), 899.

4. R. Joshi, P. Pohl, D. Strachotova, P. Herman, T. Obsil, V. Obsilova. Biophys. J., accepted (2022), DOI:

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).