Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains.
Rohit Joshi, Pavel Pohl, Dita Strachotova, Petr Herman, Tomas Obsil, Veronika Obsilova
Author Information
Rohit Joshi: Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Czech Republic; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Pavel Pohl: Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Czech Republic.
Dita Strachotova: Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic.
Petr Herman: Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic.
Tomas Obsil: Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Czech Republic; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic. Electronic address: obsil@natur.cuni.cz.
Veronika Obsilova: Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Czech Republic. Electronic address: veronika.obsilova@fgu.cas.cz.
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-labeled 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.
