Structural and biochemical characterisation of RING E3 mediated ubiquitination

Patel, Amrita (2018) Structural and biochemical characterisation of RING E3 mediated ubiquitination. PhD thesis, University of Glasgow.

Abstract

The ubiquitination pathway involves a cascade of three enzymes (E1, E2 and E3) that work together to conjugate ubiquitin (Ub) to protein substrates. RING E3s are the biggest family of E3s and are the major focus of my research project. RING E3s are characterised by the presence of a RING domain that binds E2~Ub and a protein-protein interaction domain that binds substrate(s). To facilitate Ub transfer, RING E3s perform several functions. Firstly, RING E3s bind E2~Ub and prime the E2~Ub thioester bond in a configuration that is optimal for catalysis. Secondly, RING E3s recruit substrate and juxtapose the E2~Ub thioester bond and ε-amino group of a lysine residue from the substrate to facilitate Ub transfer. Lastly, RING E3s recruit additional E2~Ub to catalyze polyUb chain formation. How RING E3s achieve these steps remains elusive. My research project focuses on understanding how RING E3s activate E2~Ub and catalyze Ub chain formation.
Previously, our lab has shown that Ub bound non-covalently to the surface of UbcH5B opposite its active site (“backside” bound Ub or UbB) stimulates ubiquitin transfer in both RING-dependent and RING-independent manners, but with a more prominent effect in RING-dependent transfer. In this earlier work, we used a monomeric RING E3, RNF38, for structural analyses. To assess whether the mechanism of UbB-stimulation is conserved for other RING E3s, I report the structure of a dimeric RING E3, cIAP1 (also known as BIRC2), bound to UbcH5B–Ub and UbB. This complex structure is the first structure of a dimeric RING E3 bound to E2–Ub and UbB. It is also the first structure of the cIAP1 RING domain bound to E2–Ub. Using structural and biochemical analysis, I show residues within the RING-UbcH5B–Ub and UbB-UbcH5B interfaces that are important in Ub transfer. I have also shown that the mechanism of UbB-stimulation is conserved throughout the UbcH5 family (UbcH5A, UbcH5B, UbcH5C and UbcH5D) of E2’s.
Previous studies suggest that UbcH6 lacks a UbB-binding ability despite sharing sequence and structural homology with the UbcH5 family. To investigate this, I performed NMR experiments that showed very weak binding between UbcH6 and Ub. To investigate further I performed autoubiquitination and lysine discharge assays using wild type UbcH6 (WT) and UbcH6 with Ser68 mutated to Arg to abrogate backside binding and found that Ub transfer was slower with UbcH6 S68R. Together my results show that UbcH6 binds Ub non-covalently via its backside, but the binding is weak compared to the UbcH5 family of E2s.
Finally in chapter 5, I describe my attempts to elucidate how RING E3s promote Ub transfer from E2~Ub onto a lysine residue of substrate. I developed a model system containing an E3 bound to E2~Ub and a monoubiquitinated substrate mimetic. Initially, I used Cbl and UbcH5B for my model since many Cbl structures have been determined and Cbl’s substrates are also well characterised. In the beginning, generating monoubiquitinated substrate mimetics was challenging, but later I standardised a method and showed that my substrate mimetics were functional in vitro. I then set up crystallisation experiments with various combinations of E3-E2–Ub and substrate mimetics, but unfortunately all my crystals diffracted poorly. After many attempts to improve the diffraction quality of my crystals, I did not manage to get data of sufficient resolution to determine a complex structure.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Huang, Prof. Danny
• Date of Award: 2018
• Depositing User: Mrs Amrita Sanjay Patel
• Unique ID: glathesis:2018-9025
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 09 May 2018 11:04
• Last Modified: 04 Oct 2019 13:05
• URI: https://theses.gla.ac.uk/id/eprint/9025