Nedd4-2 (neuronal precursor cell-expressed developmentally down-regulated 4-2) ubiquitin ligase is one member among the nine human HECT E3 ubiquitin ligases. It is the last enzyme of the ubiquitination cascade and directly transfers ubiquitin molecules to its various substrates, therefore designating them for either endocytosis or proteasomal degradation. In accordance with its ubiquitous nature, Nedd4-2 has a variety of targets. This explains the necessity of studying its regulation since any disfunction disrupts signalling pathways and leads to the development of different pathophysiological conditions – mostly known and described is Liddle syndrome (form of hypertension resulting from electrolytic imbalance). Like all the members of HECT family, Nedd4-2 also contains three distinct domains: N-terminal C2 domain, 4 WW domains and a catalytic HECT domain. Up until now, several mechanisms of Nedd4-2 regulation were described in literature. Most notable are: autoinhibition (C2 binds to HECT) [1], regulation by calcium ions [2] and interaction with other binding partners such as the 14-3-3 dimer (associates with phosphorylated residues surrounding the WW2 domain and blocks interaction with substrates) [3, 4]. To understand how calcium ions influence this ubiquitin ligase, we must focus our attention on the C2 domain, a calcium-binding domain known to bind to membranes. It is hypothesized that its role in the ubiquitination reaction is to localize the enzyme, specifically its HECT domain near the membranous substrates (e.g., ion channels) and to be the cause of stopping the autoinhibition [2].
To determine the effect and specific binding of calcium ions to Nedd4-2, we performed two functional assays in the presence and absence of calcium: liposome-binding assay (to describe the interaction with membranes) and ubiquitination assay with fluorescently labelled ubiquitin (to describe the activity of Nedd4-2). We also wanted to see the specific interaction between the C2 domain and calcium, so we crystallized it and solved the structure. Lastly, we performed analytical ultracentrifugation to demonstrate how calcium ions influence the interaction between Nedd4-2 and 14-3-3η dimer.
This study was supported by the Czech Science Foundation (V.O., grant number: 23-04686S), the Czech Academy of Sciences (RVO: 67985823 of the Institute of Physiology) and the Visegrad Scholarship (ID number: 52310440).