Synergy of experiments and computation for investigation of multidomain protein dynamics of RBR E3 ubiquitin ligases

Abstract

Ubiquitination is a post-translational modification that regulates a multitude of cellular processes. Ubiquitin transfer to a target protein involves the sequential activity of three enzymes: E1 activating, E2 conjugating and E3 ligase enzymes. A subfamily of E3 ubiquitin ligases, RING-Between-RING (RBR) family ligases contain a conserved catalytic RBR domain consisting of three sequential subdomains, RING1, IBR and RING2, separated by exible interdomain linkers. The RING1 domain is involved in the recognition of the ubiquitin-loaded E2, while the RING2 domain contains a catalytic cysteine that forms an E3-ubiquitin thioester intermediate before the final ubiquitin transfer onto the substrate.

RBR E3 ligase family members are involved in regulating a multitude of cellular events, including cellular proliferation, developmental processes and regulation of immune signalling. The ubiquitin transfer reaction catalysed by RBR E3s is a highly dynamic process and there is little information regarding the occurring conformational changes when ubiquitin is transferred from E2 to the E3 and finally onto the substrate. It is believed that the inter-domain linkers of the RBR domain are crucial for orchestrating subdomain motion required for the ubiquitin transfer reaction.

The work presented in this thesis provides insights into the conformational dynamics of two RBR E3 ligases, HOIP and RNF144A, and how such dynamics could regulate the ubiquitin transfer reaction. The conformational preferences of the HOIP RBR domain in the apo state and in the presence of the ubiquitin-loaded E2 are determined using integrative modelling approach. Furthermore, the roles of interdomain linkers are investigated using biochemical, biophysical and modelling methods. In parallel, the dynamic behaviour of the RNF144A RBR domain is characterised using solution biophysical experiments, specifically SAXS and NMR measurements.

The study of these two distinct members of RBR E3 ligases allowed us to uncover shared commonalities and identify potential evolutionary conserved structural and functional features of RBR E3 ligases.

Date of Award	1 Jan 2021
Original language	English
Awarding Institution	King's College London
Supervisor	Katrin Rittinger (Supervisor) & Franca Fraternali (Supervisor)