Structure of UBE2K\u2013Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension

Danny T Huang,Mads Gabrielsen,Brian O Smith,Gary J Sibbet,Mark A Nakasone,Karolina A Majorek

doi:10.1038/s41589-021-00952-x

Danny T Huang, Mads Gabrielsen + Show 4 more

Open Access

https://doi.org/10.1038/s41589-021-00952-x

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Abstract

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain, to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains. This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K.

Highlights

Ubiquitin (Ub) chain types govern distinct biological processes
In di-Ub synthesis (Fig. 3f), showed no enhancement in ubiquitination of SMAC-Ub3–5 and only a marginal increase in ubiquitination with SMAC-Ub5+ (Fig. 5c and Extended Data Fig. 7d). These findings suggest that the UBAM172,L198–Ub interaction accounts for the bulk of enhanced ubiquitination in the context of Ub-primed substrates
Our chemical biology approach allowed us to capture a transient state of UBE2K in complex with RING E3, UbD and acceptor K48-Ub2

Summary

Introduction

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. The structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K. While in most cases only the Ub ligases (E3s) select substrates for Ub modification[2], for RING E3s, the ubiquitin-conjugating enzymes (E2s) govern how polyUb chains are constructed by defining the internal Ub–Ub linkages[3,4]. Combining several chemical biology approaches has enabled us to determine the structure of a RING E3, RNF38, with a donor Ub (UbD) loaded on UBE2K (UBE2K–UbD) and K48-linked di-Ub in the acceptor site

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Conclusion