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 [38]. 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.
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).