Abstract
A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. Regulation of RING E3s depends on modulating their ability to stabilise the RING bound E2~ubiquitin conjugate in the activated (or closed) conformation. Here we report the structure of the Ark2C RING bound to both a regulatory ubiquitin molecule and an activated E2~ubiquitin conjugate. The structure shows that the RING domain and non-covalently bound ubiquitin molecule together make contacts that stabilise the activated conformation of the conjugate, revealing why ubiquitin is a key regulator of Ark2C activity. We also identify a charged loop N-terminal to the RING domain that enhances activity by interacting with both the regulatory ubiquitin and ubiquitin conjugated to the E2. In addition, the structure suggests how Lys48-linked ubiquitin chains might be assembled by Ark2C and UbcH5b. Together this study identifies features common to RING E3s, as well elements that are unique to Ark2C and related E3s, which enhance assembly of ubiquitin chains.
Highlights
A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade
We established that binding of ubiquitin to Ark-like RINGs significantly increases their activity, and we characterised the complex formed between the RING domain and ubiquitin[23]
Guided by the structure of UbArk2C residues 1–73 (UbR) bound to the RING domain we prepared a fusion protein in which ubiquitin was linked to the N-terminus of the RING domain of Ark2C (Fig. 1b and Supplementary Fig. 1b)
Summary
A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. The structure shows that the RING domain and non-covalently bound ubiquitin molecule together make contacts that stabilise the activated conformation of the conjugate, revealing why ubiquitin is a key regulator of Ark2C activity. 1234567890():,; Attachment of ubiquitin to proteins is a key mechanism that determines the timing and strength of many cellular responses[1] This is because the addition of ubiquitin (ubiquitylation) controls protein longevity, abundance, and function[2]. Activation of transfer by the E3 relies on the ability of the RING domain, together with adjacent residues, to bind the E2 conjugated ubiquitin (referred to as the donor ubiquitin, or UbD) and restrict its position (Fig. 1a). Interactions between the E3 and UbD, regulate ubiquitin transfer, and this has prompted a focus on identifying the features in E3 ligases that stabilise the activated conformation of RING bound E2~Ub conjugates
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