K29-Selective Ubiquitin Binding Domain Reveals Structural Basis of Specificity and Heterotypic Nature of K29 Polyubiquitin

Abstract

SummaryPolyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.

Ubiquitin chain diversity at a glance

“…K29-polyubiquitylation of Axin disrupts its interaction with the Wnt coreceptors LRP5 and LRP6 (LRP5/6), which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling (Fei et al, 2013). Interestingly, the OTU family DUB Trabid (also known in mammals as ZRANB1), which is a positive regulator of Wnt-induced transcription, shows a strong preference for K29 and K33 linkages (Kristariyanto et al, 2015a;Licchesi et al, 2012;Mevissen et al, 2013;Michel et al, 2015;Virdee et al, 2010). Trabid has been suggested to interact with and to deubiquitylate APC (Tran et al, 2008).…”

A Highly Efficient Synthesis of Polyubiquitin Chains

“…In vitro Ub enzyme systems were first developed for the preparation of polyUbs, and their utility has been demonstrated by the synthesis of almost all types of Ub chains 15, 28, 29, 30, 31. However, K27‐polyUbs and heterogeneous polyUbs have yet to be accessed with this technology 32.…”

Make Them, Break Them, and Catch Them: Studying Rare Ubiquitin Chains

“…Two studies in this issue, Kristariyanto et al (2015) and Michel et al (2015), describe innovative ways to produce large quantities of atypical K29 and K33 ubiquitin chains and report structures and mechanisms of chain-specific recognition.…”

“…We do know a great deal about K48 and K63 chains, and details are emerging on K11 and linear chains, but knowledge of other chain types is lagging behind. In this issue of Molecular Cell, Kulathu and colleagues (Kristariyanto et al, 2015) and Komander and colleagues (Michel et al, 2015) focus the attention on K29 and K33 chains.…”

“…There are no good chain-specific antibodies, no way of recognizing, labeling or binding them, and no way of making large amounts of atypical ubiquitin chains in isolation for in vitro studies. The work of Kristariyanto et al and Michel et al provides us with new tools to do just that, and they use these tools to provide a first glimpse into the biology of rare ubiquitin chains (Kristariyanto et al, 2015;Michel et al, 2015).…”