Human neuronal precursor cell-expressed developmentally down-regulated 4-2 (Nedd4-2) ubiquitin ligase is a ubiquitous member of the family of HECT ubiquitin ligases that targets and selects different proteins for endocytosis and/or proteasome degradation. Its targets are mostly integral membrane proteins – ion channels or receptors. Any defects in this enzyme cause the development of different pathophysiological conditions and among them mostly investigated are Liddle syndrome, electrolytic imbalance, hypertension, respiratory distress, cardiovascular conditions and cystic fibrosis. This HECT ubiquitin ligase has three different types of domains: N-terminal C2 domain, four WW domains and the C-terminal HECT domain, together connected with unstructured flexible regions. Described manners of regulation have been cytosolic levels of calcium and 14-3-3 proteins, but the specific mechanism and circumstances are not completely clear. In the phosphorylated state, the modified amino acids of Nedd4-2 act as a binding site to different inhibitors and activators. In our previous research, we found that in the case of the adaptor protein 14-3-3, the phosphorylated residues are pSer342, pThr367 and pSer448, all of which are surrounding the WW2 domain [1]. When Nedd4-2 is forming the complex with 14-3-3, WW3 and WW4 domains are more rigidly bound, while the WW1 domain (adjacent to the C2 domain) and the HECT domain have shown increased mobility, which may facilitate catalytic activity [2].
The neglected part of this enzyme is the C2 domain, a domain activated by binding cytosolic calcium ions and responsible for binding to different lipids, for example those present in the cell membrane or vesicles. In that way, this interaction enables the other parts of this protein, especially the WW domains, to get near to different target molecules. What our focus was in this study is to demonstrate how 14-3-3 has an inhibitory effect on the function of C2 domain, by using the hydrogen-deuterium exchange and liposome-binding assays. Additionally, by combining aforementioned results with the data obtained from analytical ultracentrifugation, small angle X-ray scattering and mass photometry, we were also able to on some basic level structurally characterize the mechanism of inhibition. In total, this is proof that 14-3-3 has another way to influence not only the activity but also the subcellular localization of this protein. What we hope to achieve next is to try to switch the focus on only structural characterization of this complex, by either crystallography or cryo-EM, which would provide a better and more refined understanding of its regulation.
This study was supported by the Czech Science Foundation (Project 23-04686S), the Czech Academy of Sciences (Research Project RVO: 67985823 of the Institute of Physiology).