Post-translational modifications by ubiquitination are important for conserved roles in the regulation of membrane proteins. Nedd4-2, an ubiquitin ligase (E3) of the HECT family, targets membrane proteins such as ion channels and transporters for ubiquitination [1,2]. 14-3-3 proteins, a family of the conserved regulatory molecule, negatively regulates Nedd4-2 through phosphorylation by PKA. This regulation is performed by providing scaffolding for Nedd4-2, thereby preventing the interaction with Nedd4-2 and other membrane proteins. Though this is known, the molecular mechanism of this regulation largely remains unknown and is under scientific scrutiny [3, 4].
We aim to understand the structural and functional basis of 14-3-3 mediated regulation of Nedd4-2. Nedd4-2 consists typically of three domains: C2 domain, WW domain and HECT domain. Possible mechanism of the 14-3-3 mediated inhibition of pNedd4-2 includes stabilization of inactive conformation of Nedd4-2 in which, HECT and C2 domains are involved in the intramolecular interaction and steric masking of WW domains surfaces. To test this hypothesis, we plan to perform the time resolved fluorescence spectroscopy measurements using phosphorylated Nedd4-2 variants labelled by extrinsic fluorophore and monitor their interaction with 14-3-3 protein. Fluorescence spectroscopy will provide basic information on the dynamics of the interaction between Nedd4-2 ligase and 14-3-3 protein. Therefore, we prepared seven Nedd4-2 protein variants with the single amino acid cysteine, which will be fluorescently labelled, monitoring different WW domains of Nedd4-2. The position selected for cysteine variants in Nedd4-2190-581; WW1: C209, C218; WW2: C389, C414: WW3: C508, C522; WW4: C571. Measuring of rotational correlation time and determination of the mean lifetime values of excited fluorophore in Nedd4-2 alone and in the complex with 14-3-3 protein (containing no Cys residues) will allow us to trace the microenvironment of one particular cysteine amino acid, which is located at different positions within Nedd4-2 construct. Expression and purification of selected cysteine mutants has been successfully performed using a bacterial expression system.
We also crystallized the complex of 14-3-3γΔC with the peptide containing phosphorylated Ser342, solved its structure using molecular replacement and refined it at 1.61 Å resolution.
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).