AbstractThe Cullin-RING E3 ligases (CRLs) are the largest subfamily of E3 ligases that participate in many biological processes determining the fate of proteins by catalysing ubiquitin transfer to specific substrates for proteasomal degradation. SOCS2 is a component of the multisubunit CRL E3 complex (CRL5SOCS2). SOCS2 plays important roles in several cancers and is involved in diabetes and inflammatory diseases. This work aims to understand the substrate recognition mechanism of SOCS2 at an atomic level and provides structural insights to guide the development of small-molecule tools and potential drug leads.
Current structural information on SOCS2 is limited to apo form (no ligand bound). In the first part of the work, two novel SOCS2-ElonginB-ElonginC (SBC) structures in complex with substrate peptides of growth hormone receptor (GHR) and erythropoietin receptor (EpoR) were solved by X-ray crystallography with a goal to elucidate the SOCS2 recognition mechanism. Different interactions of the peptides were observed in the structures as a consequence of divergences in the peptide sequences, revealing residues required to catch specific interactions and a protein loop rearrangement as a result of the binding event. An alanine scanning of substrate peptides allowed cross-validation of the structures and identified critical interactions. Based on the crystal structure, five residues that interact with GHR were selected for which single-nucleotide polymorphisms (SNPs) are known in cancer. The results show that the SNPs mutants of SOCS2 located at the phosphotyrosine (pY) pocket are highly disruptive and abolish substrate recognition, suggesting a significant impact to SOCS2 mediated interactions.
The second and third part of the work focused on the ligand development at the pY pocket of SOCS2 SH2 domain using a combination of X-ray crystallography and biophysical techniques. Novel crystal structures of SBC in complex with pY and pY analogues were obtained, providing a starting point for compound design. A screening cascade consisting of nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was established and validated by an in-house small library of pY analogues. This workflow will facilitate the process of ligand characterisation and design towards a potent binder.
The findings of this work unravel interactions of SOCS2 with its substrates in mechanistic detail. Together with the small molecule bound structures and biophysical screening assays, this work provides insights and tools to assist future ligand discovery for CRL5SOCS2.
|Date of Award||2018|
|Supervisor||Alessio Ciulli (Supervisor)|