AbstractCullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system by catalysing the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic multi-subunit complexes that control the fate of many proteins in cells and, therefore, constitute attractive drug targets for the development of small-molecule tools and potential drug leads, such as inhibitors and chemical inducers of protein degradation.
This work presents the first crystal structure of the pentameric human CRL2VHL complex, composed of Cul2, Rbx1, Elongin B (EloB), Elongin C (EloC) and pVHL. The structure presents a closed state of full-length Cul2 and a new conformation of Rbx1, thought to be in a trajectory from inactive to active state. The thermodynamic signature of the interaction between Cul2 and pVHL-EloBC (VBC) was determined as well as mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. In addition, this work focused on an extensive approach to probe the VBC surface with peptides. A first methodology involved the structure-based design aimed at targeting the Cul2 VBC interaction. Three peptides have been shown to bind at the Cul2 binding site on EloC, however, with very weak affinities that could not be optimised, suggesting its poor ligandability. The second methodology included an unbiased screening using phage display libraries of bicyclic peptides. The screening campaign yielded peptide hits but their physicochemical properties, especially poor solubility, constituted an obstacle to the progress toward a biophysical characterisation of their binding to VBC and the development into high-affinity probes.
The findings of this work provide structural and biophysical contributions into the whole CRL2VHL complex assembly and functioning and provide insights and tools that could aid future targeting of this CRL.
|Date of Award||2017|
|Sponsors||Fundação para a Ciência e a Tecnologia|
|Supervisor||Alessio Ciulli (Supervisor)|