51HIGHLIGHTS 2020

Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity, P.D. Mabbitt (a), A. Loreto (b), M.-A. Déry (a), A.J. Fletcher (a), M. Stanley (c), K.-C. Pao (a), N.T. Wood (a), M.P. Coleman (b,d) and S. Virdee (a), Nat.

Chem. Biol. 16, 1227-1236 (2020); https:// doi.org/10.1038/s41589-020-0598-6. (a) MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee (UK) (b) John van Geest Centre for Brain Repair,

University of Cambridge (UK) (c) Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee (UK) (d) The Babraham Institute, Babraham Research Campus, Cambridge (UK)

[1] K.C. Pao et al., Nature 556 (7701), 381-385 (2018). [2] M.P. Coleman & A. Hoke, Nat. Rev. Neurosci. 21(4), 183-196 (2020). [3] K.C. Pao et al., Nat. Chem. Biol. 12(5), 324-31 (2016). [4] P.D Mabbitt et al., Nat. Chem. Biol. 16(11), 1227-1236 (2020).

PRINCIPAL PUBLICATION AND AUTHORS

REFERENCES

Fig. 37: a) Crystal structure of the RCR-E2-Ub complex. E2 (mauve) and Ub (grey) are shown as cartoons. The RCR domain is shown in surface representation; RING domain (blue), linker helix (purple), helix-turn-helix (green), TC domain (orange). The trapped tetrahedral ubiquitin transfer intermediate and Trz moiety are depicted as balls and sticks. b) Close-up of the transfer intermediate.

E3s, in part because it is difficult to crystallise complexes of weakly associated proteins. The RCR-E2-ubiquitin complex is also highly transient, so in order to capture the complex, a chemically engineered E2-ubiquitin conjugate was employed that acts as an activity-based probe (ABP) for cysteine-dependent E3 ligases [3,4] (Figure 36).

The three-way complex was crystallised and X-ray diffraction data were collected at beamline ID29. The crystal structure revealed that the site containing the upstream cysteine, which is disordered in the apo-structure, forms a short- alpha helix upon E2-Ub binding (Figure 37). Furthermore, when bound to the RCR, the E2- Ub conjugate adopts a conformation with an intermediate state of activation, which tempers its intrinsic reactivity toward lysine substrates. This likely prevents transfer of ubiquitin to lysine substrates while promoting productive ubiquitin relay. The transient helical ordering of the upstream site upon E2-E3 transfer might act like an entropic spring , thereby providing the

driving force for the relay process. It is unclear if this mechanism is unique to MYCBP2 or if there are other E3s that use an RCR-like mechanism.

Biochemical experiments suggest that ubiquitin relay is a robust process and that the residues flanking the upstream cysteine have little or no effect on relay. This makes bioinformatic searches for other E3s with RCR-like activity difficult and further emphasises the utility of activity-based probes for E3 discovery. To probe the RCR mechanism and non-lysine ubiquitination in mammalian neurons, mice were gene-edited so that the upstream cysteine was mutated to alanine. Neurites cultured from these mice displayed demonstrated increased tolerance to injury, consistent with a biological role for E2-to-RCR ubiquitin transfer and subsequent ubiquitin relay to threonine residues within substrates. Excitingly, the results suggest that a specific inhibitor of this seemingly unique RCR mechanism may have therapeutic value for the treatment of a range neurological disorders and neurodegenerative diseases.