Researchers in the laboratory of Professor Alessio Ciulli have revealed atomic-resolution images of how an E3 ubiquitin ligase enzyme involved in immune and cell signalling latches on to its protein partners. This is important because these binding events are required for the protein to function properly inside the cell.
E3 ubiquitin ligases act on specific substrates in cells, naturally tagging them with a protein called ubiquitin. This in turns leads to changes in protein function and stability, such as inducing intracellular protein degradation via the cell’s proteasomes. There are estimated to be over 600 E3 ligases in the human cell, and many are involved in human diseases, providing a plethora of attractive targets for drug discovery. However, in many cases little is known about how E3 ligases recognize natural substrates at the molecular level.
In their work, published today in Nature Communications, PhD student Wei-Wei Kung, working alongside postdoctoral researchers Dr Sarath Ramachandran and Dr Nikolai Makukhin, now shine light on a new E3 ligase called suppressor of cytokine signaling 2 (SOCS2). SOCS2 is a component of the large family of Cullin5 E3 ubiquitin ligase complexes, and a key player in cell signalling. Using protein X-ray crystallography, the authors determined the first cocrystal structures of SOCS2 in complex with phosphorylated peptides from two of its substrates: the growth hormone receptor (GHR) and erythropoietin receptor (ER), which are important signalling proteins located at the plasma membrane.
“There is growing interest in understanding and targeting E3 ubiquitin ligase proteins for drug discovery,” explained Ciulli. “This is because of the potential to design molecules that hijack E3 ubiquitin ligases and re-directed them towards unwanted disease-causing proteins as a means to target them for degradation. However, only 3 out of the ~600 known E3 ligases are being targeted using this approach. To expand the reach to new E3 ligases, a first step is to elucidate how E3 ligases bind to their natural substrates, which also helps to understand how they work.”
The new crystal structures, solved by Wei-Wei and Sarath, show how SOCS2 grabs the GHR and EpoR substrates by docking them to specific tyrosine residues as these become phosphorylated. Additional biophysical studies conducted by Nikolai and Wei-Wei further characterized the binding affinities and the role of cancer-causing mutations on SOCS2.
“Shining light at atomic detail on protein interactions provides information-rich start points for drug design”, explain Nikolai and Sarath. “The insights we have revealed in this work will seed the development of novel small-molecule ligands for SOCS2. Such ligands could be used as SOCS2 inhibitors in their own right, and as part of new heterobifunctional degrader molecules also called Proteolysis-targeting chimeras (PROTACs), to target proteins of interest to degradation”.
This work, which was funded by the European Research Council, therefore not only reveals the molecular basis for substrate recognition by SOCS2, but also lays the foundation for future studies to design new small molecules targeting this biologically important E3 ligase.