Initial structural studies of regulation of ubiquitin ligase Nedd4-2 by 14-3-3 proteins

P. Pohl1,2, T. Obsil1,3 a V. Obsilova1

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

22nd Faculty of Medicine, Charles University, 150 06 Prague 5, Czech Republic

3Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague 2, Czech Republic

pavel.pohl@fgu.cas.cz

 

14-3-3 proteins belong to evolutionarily highly conserved family of regulatory proteins which regulate a variety of biological processes by binding to specific phosphorylated motif of their binding partners [1]. One of several hundreds of 14-3-3 protein binding partners is ubiquitin ligase Nedd4-2 (NEDD4L), whose role is ubiquitination of various ion channels and membrane transporters [2]. The best-described example is the regulation of the epithelial sodium channel (ENaC), which with its activity in the distal renal tubule, contributes to maintaining Na+ homeostasis of the whole organism. Mutations of Nedd4-2 gene are associated with developmental disorders, hypertension and epilepsy. Dysregulation of Nedd4-2 in mice also leads to respiratory, renal, cardiac, and neural disorders and negatively affects the immune system [3–8]. Phosphorylation of specific serine and/or threonine residues allows the binding of 14-3-3 proteins, which results in the inhibition of interaction between Nedd4-2 and its substrate [9].  However, the structural nature of the mechanism of regulation by 14-3-3 protein has not been elucidated yet.

We have expressed and purified stable constructs of Nedd4-2 ligase and we confirmed its interaction with 14-3-3 protein in phosphorylation-dependent manner. We also identified the amino acids involved in the interaction between these molecules and performed initial biophysical characterization using analytical ultracentrifugation and small angle X-ray scattering. Our results show that: Nedd4-2186-975 is monomeric and the stoichiometry of Nedd4-2186-975:14-3-3h complex is 1:2 (with possible shift to 2:2 in high concentrations). The fluorescence polarization assays with short peptides of Nedd4-2 containing single 14-3-3 binding motif, in combination with site-directed mutagenesis and native TBE electrophoresis, show, that the key Nedd4-2 residues important for the interaction with 14-3-3 proteins are phosphorylated Ser342 and Ser448. We also crystallized the complex of 14-3-3γΔC with the peptide containing phosphorylated Ser448 and solved its structure using molecular replacement and refined it at 1.73 Å resolution.

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This study was supported by the Czech Science Foundation (Project 20-00058S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and by Grant Agency of Charles University (Project 740119).