PhD student - Laboratory of Structural biology of signaling proteins

Nedd4-2 is a HECT E3 ligase that plays a critical role in pathophysiology by regulating multiple substrates, including the epithelial sodium channel (ENaC). Pathological consequences of Nedd4-2 dysregulation include respiratory distress, hypertension, electrolyte imbalance, and kidney disease. In addition to the not yet fully elucidated Ca2+ - dependent regulation of Nedd4-2, Nedd4-2 is regulated via phosphorylation and binding to various binding partners including scaffolding proteins 14-3-3. 14-3-3 proteins have the ability of binding the functionally different signal proteins, including kinases, phosphatases and transmembrane receptors by changing their function. The aim of this project is to elucidate the regulatory mechanism human (Smurf1, Nedd4L), yeast (Rsp5) HECT E3 ubiquitin ligases and to understand the role protein-protein interactions in their regulation.

For this purpose, the structure of Nedd4L, Smurf1, Rsp5 complexes with different binding partners (ClC-5, 14-3-3 proteins, arrestins) will be studied using integrative structural biology methods, in particular cryo-EM, SAXS, H/D exchange coupled to MS and chemical crosslinking coupled to MS. The structure of these complexes will be solved by combining the results of experimental methods and methods of structural bioinformatics (if needed). The activity of E3 ligases will be measured using liposome-binding, pulse-chase and autoubiquitination assays. Combined, these approaches will allow us to elucidate regulatory mechanisms of Nedd4-like E3 ubiquitin ligases. This is a project in the field of structural biology and  biophysical chemistry of proteins and is funded by grants from the research group. Cooperation with the Faculty of Science, Charles University in Prague.