Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.

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Proteomics Proteomics

Mechanisms of ubiquitin recognition and their influence on the application of ubiquitinbinding domains as tools to investigate ubiquitin-modified proteomes

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Daniel Scott , Neil J Oldham , Jo Strachan , Mark S. Searle , Robert Layfield *

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School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK

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School of Chemistry, University Park, University of Nottingham, Nottingham NG7 2RD, UK

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School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

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Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD,

UK

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Corresponding author:

Dr Robert Layfield Tel +44-115-823 0107 Fax +44-115-823 0142 [email protected]

Keywords: synthetic biology; ubiquitin; ubiquitin-binding domain

Word count: 11754

Received: 21-Jul-2014; Revised:05-Sep-2014; Accepted: 13-Oct-2014

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/pmic.201400341. This article is protected by copyright. All rights reserved.

Abbreviations (non-standard)

DSB - DNA double strand breaks DUB – deubiquitinating enzyme ERAD - ER-associated degradation LUBAC - linear ubiquitin chain assembly complex NZF - Npl4 zinc finger PSAQ - protein standard absolute quantification TUBEs - Tandem Ubiquitin-Binding Entities UBA - ubiquitin-associated (domain) UBD - ubiquitin-binding domain UiFC - ubiquitination-induced fluorescence complementation UIM - ubiquitin-interacting motif ZnF - zinc finger

This article is protected by copyright. All rights reserved. Page | 2

Abstract Ubiquitin-binding domains (UBDs) are modular units found within ubiquitin-binding proteins that mediate the non-covalent recognition of (poly)ubiquitin modifications. A variety of mechanisms are employed in vivo to achieve polyubiquitin linkage and chain length selectivity by UBDs, the structural basis of which have in some instances been determined. Here we review current knowledge related to ubiquitin recognition mechanisms at the molecular level and explore how such information has been exploited in the design and application of UBDs in isolation or artificially arranged in tandem as tools to investigate ubiquitin-modified proteomes. Specifically we focus on the use of UBDs to directly purify or detect (poly)ubiquitin-modified proteins and more broadly for the targeted manipulation of ubiquitinmediated processes, highlighting insights into ubiquitin signalling that have been provided. 1. Introduction and historical perspectives 1.1 Protein PTM by ubiquitin Reversible PTM of proteins allows cells to respond to external and internal stimuli to control intracellular signalling events. Ubiquitin is a small protein, which functions as a covalent modifier of other target proteins, in order to control a whole array of cellular processes including protein degradation, signal transduction and DNA repair pathways [1]. Modification typically involves isopeptide bond formation between ubiquitin’s C-terminal Gly76 and specific lysine residues within the target protein sequence, catalysed by a series of enzymes termed E1-E3. Ubiquitin itself also contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) as well as an N-terminal methionine (Met1), which can each act as modification sites (Figure 1A), such that polyubiquitin chains containing multiple ubiquitin moieties can be assembled on target proteins (Figure 1B). Like other PTMs, ubiquitination is reversible and a family of deubiquitinating enzymes (DUBs) mediate the regulated removal of ubiquitin modifications in balance with the general E3 ligase activity (Figure 1C) [2]. Polyubiquitin chains represent a versatile, three-dimensional code [2] in which different linkages (involving different lysine residues) within the chains give rise to varying topologies that present different recognition and binding surfaces [1] (Figure 2). Eight types of homogenous chains containing all the same linkage sites are possible, as well as chains of different length (numbers of ubiquitin moieties). As an added level of complexity, mixed linkage polyubiquitin chains have been reported e.g.


involving more than a single lysine residue within the chain, as well as branched chains where a single ubiquitin within a chain is modified at two or more sites (Figure 1B) [3]. The physiological significance of such complex modifications and, for example, mechanisms explaining their apparent ability of the latter to further enhance recognition by proteasomal ubiquitin receptors and rates of substrate degradation [3] are only just beginning to emerge. Our understanding of the molecular basis for deconvoluting these different signals and the specific in vivo functions of this extensive and diverse pool of multiply linked protein polymers is still in its infancy.

1.2 Ubiquitin-binding proteins and ubiquitin-binding domains The ubiquitin code is ‘read’ by more than 20 different families of ubiquitin-binding domains (UBDs; Table I) within intracellular ubiquitin-binding proteins and ubiquitin receptors [4] that participate in non-covalent interactions with the different ubiquitin surfaces presented by these modifications. From a historical perspective, one of the first ubiquitin-binding proteins to be characterised was a component of the 26S proteasome, a multi-subunit protease capable of recognising and degrading certain ubiquitin-modified substrates. A 50kDa subunit of the 26S proteasome, S5a/RPN10, was initially shown to bind efficiently to polyubiquitin chains containing at least four ubiquitins [5, 6]. Mammalian S5a was found to contain two independent polyubiquitin-binding sites [7], which were established to be simple motifs. Bioinformatic analyses uncovered similar sequences in other proteins, which subsequently came to be defined as ubiquitin-interacting motifs (UIMs) [8], now known to represent short single amphipathic helices (Figure 5a). Hence UIMs are the archetypal UBDs. Many more UBD families have now been identified, of both simple and complex structures (Figures 3, 5; Table I) [1].

1.3 Applications of UBDs as in vitro tools The binding selectivities of UBDs for (poly)ubiquitin, and the covalent and stable nature of the PTM itself, has led to various attempts to exploit UBDs in the affinity purification of ubiquitin-modified targets. These initially involved using complete ubiquitin-binding proteins (including their composite UBD sequences) [9] followed by simple UBDs in isolation [10] and artificial protein constructs containing tandem arrays of UBDs [11]. UBD sequences with polyubiquitin-selectivity have also more


recently been developed as probes to monitor dynamic changes in ubiquitination patterns in live cells [12] as well as to specifically manipulate components of the ubiquitin system [13]. In this article we specifically review the uses of UBDs as research tools and speculate on future prospects and challenges in these areas. In order to give a clearer insight in to the development of UBDs as research tools, we first provide an overview of ubiquitin recognition mechanisms by UBDs at the molecular level.

2. Endogenous mechanisms which underlie the selectivity of (poly)ubiquitin recognition 2.1 The molecular basis of ubiquitin recognition by UBDs An Ile44-centred hydrophobic face on ubiquitin is commonly targeted by the non-covalent interaction of UBDs (including the UIM), with some variation in the surrounding residues recognised by different UBDs. Further, an acidic hydrophilic Asp58-centred face, as well as residues at the extreme C-terminus of ubiquitin (when not conjugated to targets) also represent UBD recognition sites (Figure 2) [4]. Although these defined binding patches on the surface of ubiquitin are apparent, interactions at these sites are by no means mutually exclusive and allow for multiple UBDs (from the same or different ubiquitin-binding proteins) to simultaneously interact with a single ubiquitin moiety [14]. For example the ubiquitin-associated (UBA) domain of the p62 protein and the A20 zinc finger (ZnF) domain of the ZNF216 protein, UBDs which target the Ile44- and Asp58-centred faces respectively, are able to bind unhindered on a single ubiquitin [14] (Figure 2A). A more systematic study of members of other UBD families indicates that although there is considerable overlap of binding sites, reflecting the predominance of Ile44-binding UBDs, many other opportunities exist for multiple interactions and ubiquitin-mediated co-localisation of UBDs and their parent ubiquitin-binding proteins. The modelling is supported by a number of X-ray structures; three moieties of the A20 ZnF4 domain of the A20 protein (see section 2.3) are able to bind in different orientations to three unique surface patches of a single ubiquitin [15] (Figure 3A), while the Rabex5 nucleotide exchange factor shows UBDs simultaneously interacting with two sites (Ile44 and Asp58) on a single ubiquitin molecule (Figure 3B) [16, 17].

The majority of UBDs bind to monomeric ubiquitin through surprisingly weak interactions, typically in the range 10-500 M. However, multiple low affinity interactions involving tandem UBDs (see section


2.4) are the basis for much higher affinity binding (range 1-15 M) to polyubiquitin chains driven by avidity effects [18]. Since cytosolic concentrations of ubiquitin are thought to be in the low micromolar range [6, 19], avidity effects are critical for fine tuning of signalling events and avoiding non-specific binding to this ubiquitin pool. Multiple UBDs placed in tandem within the same primary sequence, or multiple UBDs from different ubiquitin-binding proteins brought together in a complex, can interact simultaneously with different ubiquitin moieties to realise avidity effects that enhance binding affinity and contribute to polyubiquitin chain linkage selectivity [1]. As an alternative, in some cases two binding surfaces on the same UBD can be used to simultaneously interact with two different ubiquitin moieties linked within a polyubiquitin chain e.g. the UBA2 domain of the proteasome shuttling factor hHR23A [20] (see section 2.3) and similarly the UBA domain of the S. pombe protein Mud1 [21]. In contrast, protein dimerization can lead to two UBA domains being displayed to increase the affinity for longer polyubiquitin chains, as observed for the dimeric E3 ligase Cbl-b. [22]. A variation of these themes is the co-localisation of different ubiquitin-binding proteins on the same polyubiquitin chain by employing hybrid chains containing different linkage types, as evident in the IKK signalling network by co-localising two different kinase complexes in activating the innate immune response mechanisms [23].

The prevailing model is that the structurally unique (poly)ubiquitin modifications can recruit different effector proteins (ubiquitin-binding proteins) via interactions with their UBDs, to influence the fate of the modified protein. Essentially polyubiquitin chains of varying topologies present their UBD interaction surfaces within a defined region of conformational space, with individual UBD-containing proteins and complexes selecting conformations sampled by these interaction surfaces that may be unique to different chain topologies. As intimated above, polyubiquitin chain selective binding by UBDs can be achieved by various mechanisms including the use of tandem UBDs positioned in such a way to favour multivalent interactions with specific chain topologies, and recognition of sequences adjacent to isopeptide linkages by individual UBDs with multiple binding surfaces. In some cases the detailed molecular and structural basis for polyubiquitin selectivity has been determined, in particular as a result


of efforts from the Dikic and Komander labs, and selected examples including several relevant to research applications of UBDs are highlighted in the following sections.

2.2 Polyubiquitin chain topologies Insights into the structures of in vitro-generated polyubiquitin chains in solution by NMR, in X-ray structures and from single-molecule FRET analysis have provided a useful reference point for understanding the basis of linkage-dependent selectivity in ubiquitin signalling. Whilst the structural variation is considerable amongst the various diubiquitins (the minimum units of polyubiquitin chains) so far characterised, varying from compact or closed conformations (Lys48-, Lys11- and Lys6-linked) where some UBD binding sites appear to be occluded, to open extended structures (Lys63 and linear Met1-Gly76), the more or less flexible nature of the covalent linkage means that each linkage type is, however, likely to be associated with an ensemble of dynamic structures in conformational exchange (Figure 4A). NMR studies of Lys48-linked diubiquitin reveal that the Ile44 binding surfaces of both distal and proximal ubiquitin motifs form weak contacts with an equilibrium constant between open -1

and closed forms of ca. 6 M [20, 24]. In contrast, Lys11-linked diubiquitin also forms a compact structure (interface), evident in X-ray structures and in solution by NMR, in which the Ile44 site is fully exposed [25]. Moreover, this intrinsic adaptability appears to be an important factor in accommodating binding partners. As a result, ligand binding leads to either extensive chain remodelling or selection of pre-existing conformations [26] such that different UBDs can in turn populate an ensemble of bound conformations for the same polyubiquitin chain type. To illustrate this point, in a recent review [27] an overlay of complexes of Lys63-linked diubiquitin with a selection of different UBDs reveals considerable divergence in the orientation of distal and proximal ubiquitin moieties in bound (UBD) complexes. This inherent malleability of polyubiquitin chains allows UBDs to exploit, using various mechanisms, conformational selection to facilitate linkage-specific polyubiquitin chain recognition in vivo. The implications for the application of UBDs as linkage-selective tools are that a range of different UBDs can be (and have been) used to afford polyubiquitin recognition in vitro (see sections 3-6).

2.3 Polyubiquitin recognition by individual UBDs


Several isolated UBDs have been shown to use multiple binding surfaces on the same UBD unit to achieve polyubiquitin linkage selectivity. The UBA domains of hHR23A (UBA2) and Mud1 have illustrated how two surface patches on the UBD allow it to be sandwiched into the hydrophobic binding cleft formed by Lys48-diubiquitin using the Ile44 faces of both proximal and distal ubiquitin moieties (Figure 4C) [20, 21]. In contrast, most other UBA domains (including the UBA1 domain of hHR23A and UBA domains of p62, ubiquilin-1 (known as UQ1, Figure 2B), Ede1 and Dsk2) bind the Ile44 face using a single binding site, which includes a conserved M/L-G-F/Y loop motif between UBA helices 1 and 2 [28]. These show no selectivity and display a very wide range of affinities (Kd ~ 400 – 10 M). Similarly, the A20 ZnF domain (for example from the ZNF216 protein) targets the Asp58-centred face of ubiquitin in a simple binding mode (Figure 2B) but with relatively high affinity for a UBD (Kd ~ 12 M) [14]. By analogy with the UBA2 domain of hHR23A, the small A20 ZnF4 domain of the A20 protein, a polyubiquitin chain-editing enzyme, which is able to bind to and disassemble Lys63-linked polyubiquitin chains, is able to make multiple contacts with different ubiquitins within a chain (Figure 5B). This is achieved via the three individually weak ubiquitin-binding sites on the small A20 ZnF4, which together lead to avidity effects that are selective for tight binding to Lys63-triubiquitin, rather than Lys48-, Lys11-linked or linear chains [15]. Although one of the three different binding sites on the A20 ZnF4 motif (an inverted UIM) uses the classical Ile44 ubiquitin face, the two other patches use non-classical ubiquitinbinding sites involving the TEK box motif (Thr9-Lys11) and a loop region of ubiquitin (around residues Glu51-Lys63). As a further example, TAB2 (Figure 5C) and TAB3 proteins, which are ubiquitin-binding subunits of the TAK1 kinase complex involved in upstream events in NF-B signalling, recognise polyubiquitin chains via Npl4 zinc finger (NZF) UBDs and each has two ubiquitin-binding sites for avid ubiquitin-binding interactions [29]. Unlike the A20 ZnF4 domain, linkage selectivity appears to be much weaker, with Lys63- and Lys48-diubiquitin binding with only a three-fold difference in affinities. In contrast the less flexible Met1-linked linear ubiquitin dimer, whose accessible conformational space appears to be more restricted, shows a 150-fold lower affinity [30]. The HOIL-1L subunit is a component of the linear ubiquitin chain assembly complex (LUBAC) and also contains a NZF domain, which carries the classical zinc-coordinating core region with a second binding site within an extended helical tail region, which together mediate interactions with Met1-linked linear diubiquitin [31]. While the NZF


core recognises the Ile44 hydrophobic patch of the distal ubiquitin, it simultaneously recognises a novel Phe4 surface on the proximal ubiquitin, which also forms contacts with the NZF tail (Figure 5E). As a final example the structure of the sandwich complex of the UIM of Hrs (a component of the ESCRT-0 protein complex) illustrates how the phasing of two interaction surfaces on opposite faces of a single helix can accommodate two (in this case not covalently linked) monomeric ubiquitins with minimum steric interaction [32]. Together these examples demonstrate the versatility of just a small single UBD, and in some instances the importance of the spatial relationship of the interaction surfaces in mapping to the conformational space accessible to certain polyubiquitin chain topologies and not others. Accordingly, in some cases individual UBDs have sufficient intrinsic polyubiquitin selectivity to find utility as in vitro tools (see sections 3-6).

2.4 Polyubiquitin recognition by tandem UBDs However many ubiquitin-binding proteins contain multiple UBDs, as either repeats of the same motif or a collection of different and complementary domains either relatively rigidly linked or flexibly connected by disordered loops. As an example of a multi-UBD protein, the DUB isopeptidase T (isoT/USP5), which is responsible for the disassembly of unanchored (substrate-free) polyubiquitin chains contains a UBD known as the Znf_UBP (or BUZ) in tandem with two UBA domains [33]. The Znf_UBP targets with high binding affinity the exposed C-terminal di-Gly motif of the proximal ubiquitin (that at the end of the chain which would be substrate-linked in conjugated polyubiquitin) within an unanchored chain, which is inserted into a deep binding pocket (Figure 2) whereas the UBA domains contribute polyubiquitin chain recognition by binding to Ile44-centred patches exposed in other ubiquitins within the chain. Intuitively the relative orientation and spacing of tandem UBDs contributes to polyubiquitin chain selectivity, none more so than with the simplest of UBD, the single helix UIM. The proteasome subunit S5a/RPN10 achieves polyubiquitin selectivity via two flexibly linked UIMs that bind to the Ile44 patches on ubiquitin motifs within Lys48-linked diubiquitin (Figure 5A). NMR studies have shown degeneracy in the mode of interaction, with UIM1 and UIM2 able to interchange their interactions in binding proximal or distal ubiquitins within the Lys48-linked diubiquitin, with only a slight preference between the two possible modes [34]. Extended interactions


via UIM1 have been suggested to account for the experimental observations that both Lys48-linked triubiquitin and tetraubiquitin are recognised with higher affinity than the shorter dimer. In contrast, the DNA repair protein Rap80 uses two rigidly connected UIMs in tandem within a single extended helix to achieve avid interactions which are selective for Lys63-diubiquitin but not Lys48-linked or linear ubiquitin dimers [35]. Here the orientation and spacing of the two UIMs allows for selectivity by exploiting differences in the separation of the Ile44 binding sites for distal and proximal ubiquitin motifs in different extended diubiquitins, which uniquely favours the spacing within the Lys63-linked dimer (Figure 4B).

Although structural models of Lys63-linked and linear Met1-linked chains suggest many similarities in conformational flexibility of the extended chain, discrimination for the latter can be achieved by exploiting tandem UBD interactions in the context of a dimeric coiled-coil (contrasting with tandem UBDs embedded within the same primary sequence). The functional role of linear polyubiquitin chains is now well established (e.g. in relation to NF-B signalling pathways) and here selective recognition is achieved by the UBAN UBD found in NEMO and ABIN proteins. In the case of NEMO the X-ray structure of the helical coiled-coil complex reveals that the distal and proximal ubiquitin motifs of a linear ubiquitin dimer are recognised by motifs on different helices within the UBAN coiled-coil dimer [36]. The distal ubiquitin is recognised mainly through the canonical Ile44 patch but also Arg71, Leu72 and Arg73, however the proximal ubiquitin motif forms specific interactions via Gln2 and Glu16 at its Nterminus, thus demonstrating specific recognition of the linker region (Figure 5D). The bound conformation of linear diubiquitin is strikingly different in the complexes of NEMO UBAN and the Npl4 o

NZF of HOIL-1L (Figure 5E) with the proximal ubiquitin in the latter rotated by >90 to achieve specific contacts with the rigidly oriented UBD binding surfaces [27]. However, both complexes demonstrate selectivity by not using the Ile44 patch on the proximal ubiquitin, but rather residues (Phe4 or Gln2/Glu16) on the N-terminal -hairpin of ubiquitin close to the Met1 conjugation site. As will be discussed, many of the UBD-based tools that have been developed for the recognition of polyubiquitin modifications are, as such, bio-inspired given that they are composed of multiple UBDs (artificially) positioned in tandem.


2.5 UBD selectivity for polyubiquitin chain length How UBDs can be used as a ‘molecular ruler’, to select on the basis of polyubiquitin chain length, remains a relatively unexplored aspect of recognition studies linked to specificity in biological signalling. There are some clear examples where higher affinity interactions are detected with longer polyubiquitin chains, suggesting that avidity effects, utilising a set of individually weak UBD-ubiquitin interactions, can be employed to enhance chain linkage selectivity by out-competing weaker nonspecific interactions at the level of individual ubiquitin motifs. It is, however, unclear at what point these avidity effects are optimised for the desired signalling outcomes. Lys48-linked tetraubiquitin, amongst an increasingly diverse collection of chain types, is apparently a minimal signal for efficient proteasomal targeting of some substrates [37], whilst a Lys63-triubiquitin appears to be the minimum requirement as a potent activator of the RIG-1 kinase [38]. In the former case, sensing of polyubiquitin chain length in yeast to promote protein degradation can be achieved by dynamic rearrangements of ternary complexes involving two different ubiquitin-binding proteins, Rpn10 (S5a equivalent) and Dsk2, using their respective UIM and UBA domains, with the orientations of the complexes (and presumably proteasomal accessibility) dependent on the numbers of ubiquitins within the chain [39]. In the case of the p62 protein, a combination of UBA domain dimerisation, coupled with p62 oligomerisation via its PB1 domain, offers a further possible insight into sensitisation towards longer polyubiquitin chains [40, 41]. Here UBA domain dimerisation which competes for the same binding sites as ubiquitin diminishes the binding affinity for monomeric ubiquitin and shorter polyubiquitin chains, with avidity effects from p62 oligomerisation appearing to favour the binding to longer chains of Lys63-linked polyubiquitin that is essential for the NF-B signalling function of p62. Knowledge gaps in this area of fundamental mechanisms which regulate the selective discrimination of polyubiquitin chain length present a key challenge in synthetic biology with respect to developing useful tools and reagents.

3. The application of UBDs to the purification of ubiquitin-modified proteins Various strategies have been applied to the affinity purification of intact ubiquitin-modified proteins including the use of heterologously-expressed epitope-tagged ubiquitin [42] as well as non-selective [43]


or isopeptide linkage selective ubiquitin antibodies [44], often upstream of MS-based identifications [45, 46]. Antibodies that recognise diglycine (diGly)-containing isopeptides following trypsin digestion are also currently pursued in the field and used to directly define sites of ubiquitin modification [47] although some structural information is lost with such “bottom up” approaches. For the purposes of this review we focus on methods reliant on the use of ubiquitin-binding proteins and their composite UBDs. The various MS approaches used to identify ubiquitin-modified proteins, define modification sites, and to provide structural information on the modifications themselves are detailed elsewhere [45, 48, 49].

3.1 Use of ubiquitin-binding proteins Notably one of the first characterised ubiquitin-binding proteins, S5a/Rpn10, was also one of the first to be applied to the affinity-enrichment of ubiquitin-modified proteins in vitro. Using a recombinant GSTtagged version of full-length human S5a (containing its two tandem UIMs) immobilised on beads, mixtures of ubiquitinated proteins were purified from brain and placental tissue [9]. Several subsequent analyses have made use of the polyubiquitin-binding affinity of S5a, for example in the context of studying synapse remodelling and signalling [50], the mechanism of action of an inhibitor of anthrax lethal toxin [51], with one study making use of MS analyses to identify a number of proteins that were hyper-ubiquitinated from heart tissue taken from patients with dilated cardiac myopathy, compared to both ischaemic and unused donor hearts [52]. Furthermore in a similar manner, GST-fusions of the ubiquitin-binding proteins Rad23 and Dsk2 were utilised to screen for substrates of the 26S proteasome in budding yeast [53]. 3.2 Use of tandem UBDs A step-change in the field of studying ubiquitin PTMs came from the development of TUBEs (Tandem Ubiquitin-Binding Entities), which, inspired by arrangements found in nature, represent multiple repeats of UBD sequences engineered into the same amino acid chain [11]. A number of attempts to generate such synthetic multiple UBD constructs capable of polyubiquitin chain-selective recognition have been reported, using a range of UBD sequences. Briefly, these typically represent as many as five UBD repeats separated by flexible polyglycine linkers, and often bead-immobilised (or immobilisable) via a diverse range of epitope tags. TUBES based on the UBA1 domain of hHR23A and UQ1-UBA of


ubiquilin-1, have been reported to exhibit high affinity avid binding to Lys48- and Lys63-linked tetraubiquitin chains (100-1000 fold enhancement in Kd over single UBA domains), allowing polyubiquitinated proteins to be efficiently captured from cell extracts [11]. These two essentially nonselective Ile44-targeting UBDs are the most commonly applied in published TUBE-based enrichments although other sequences have also been used. TUBEs have been used extensively in this purification context and a summary of some of the reported studies using TUBEs is presented in Table II, along with biological insights gained. In fact reagents have been characterised (and commercialised) with reported equivalent affinities for Lys63- and Lys48-linked polyubiquitin chains, as with the hHR23A-TUBE (2), or with a preference for the former over the latter, as exemplified by the UQ1-TUBE (1). However the full polyubiquitin-binding specificity of such reagents (i.e. against all linkage types) has not been exhaustively determined, and there are major omissions in the portfolio for reagents allowing the purification of polyubiquitin modification with ‘atypical’ linkages (including Lys6, Lys11). In addition to acting as tools for the efficient purification of ubiquitin-modified proteins, TUBEs have also been shown to exert a protective effect towards proteasomal degradation and DUB activity in cell extracts [11], allowing, for example, the recovery of otherwise labile or short-lived polyubiquitin-conjugated proteins including p53 and IB (see also section 5).

3.3 Use of isolated UBDs (Ile44-directed) It is also noteworthy that individual UBDs (i.e. not in tandem) can be applied to the affinity-enrichment of ubiquitin-modified proteins. In this case the concentration of multiple UBDs covalently immobilised on a matrix similarly (to TUBEs) promotes avidity effects, although it is cautionary to note that this has the potential to result in polyubiquitin-binding specificities that may not be reflective of those observed in solution. This point is exemplified by the fact that original work, surveying the interaction properties of 30 Ile44-directed UBA domains in their GST-tagged forms, using quantitative pull-downs and surface plasmon resonance, concluded they could be sorted in to four classes including Lys48- or Lys63-linked polyubiquitin-selective binders [54]. However, a subsequent analysis clearly demonstrated that in some case the Lys63-polyubiquitin-selectivity attributed to individual UBA domains was a result of an artefact where the dimeric GST conveniently positioned two UBAs for higher affinity, avid interactions with


Lys63-linked polyubiquitin [28]. With that caveat considered, provided that polyubiquitin selectivity of the UBDs under the experimental conditions used is clearly established, individual UBDs can still find utility in studies of ubiquitin PTMs.

Indeed the isolated UBA domain from human ubiquilin-2 (UBQLN2, also known as PLIC-2), was used to capture total polyubiquitin chains from Huntington’s disease models and patient samples [10]. MSbased analyses revealed accumulation of a variety of polyubiquitin chain linkages, indicating that broader changes to ubiquitin-mediated processes than were previously recognised were occurring during disease progression. The same UBD was used to capture total polyubiquitin chains as part of a protein standard absolute quantification (PSAQ) method designed to accurately quantify cellular ubiquitin pools [19]. A further UBA domain, from the p62 protein, was used to capture and identify as many as 200 putative ubiquitinated proteins from Arabidopsis seedlings [55], extending a prior cataloguing of such targets achieved using (tandem) UBA domains from the DUB AtUBP14 (plant equivalent of IsoT/USP5) and repeated UIMs from AtRPN10 (plant S5a) [56, 57]. Note that for the purposes of this review these sequences are not considered as TUBEs since they represent naturally occurring tandem UBD sequences, as opposed to being artificially generated. Naturally occurring tandem UIM sequences from the S5a protein were also used to reveal that subunits of the 20S proteasome machinery can be ubiquitin-modified in a muscle cell line, determined using a broad spectrum DUB-based strategy to release the modified targets from the affinity resin [58]. An additional Ile44-directed UBD, the UIM of the E3 ligase ASB2a, was also used to capture ubiquitin-modified proteins from mammalian cells, which were characterised using a candidate western blotting approach [59]. In this case the short UIM sequence represented a synthetic peptide, contrasting with other isolated (and tandem) UBDs that are usually derived from recombinant (bacterially-expressed) proteins due to their larger sizes. One recent study evaluated eight different Ile44-directed UBDs in parallel in the purification and identification of ubiquitin-modified proteomes from a mouse macrophage cell line (p62-UBA, hHR23a-UBA2, NBR1-UBA, NUB1-UBA, UQ1-UBA, Dsk2-UBA, S5a-UIM, VSP9-CUE). The authors noted significant diversity in the complement of purified target proteins and their ubiquitin modifications [60], and also suggested that the UQ1-UBA and Dsk2-UBA domains exhibit the broadest


specificity in this type of analysis. Such diversity presumably represents different inherent binding affinities and specificities of the UBDs, and highlights the fact that the purified ubiquitin-modified proteome recovered is very much dependent on the UBD-based affinity resin used.

3.4 Use of isolated UBDs (Asp58-directed) In contrast to the use of Ile44-directed UBDs in the affinity enrichment of ubiquitin-modified proteins, relatively few attempts have been made to exploit Asp58-targeting UBDs, in part reflecting the fact that there are fewer examples of the latter. However the A20 Znf domain of the ZNF216 protein (Figure 2) was used to successfully affinity purify ubiquitinated proteins from skeletal muscle, revealing the existence (through its binding) of significant levels of Lys48-linked diubiquitin in vivo [48]. A subsequent study demonstrated that the diubiquitin was comprised not only of unanchored dimers with a free distal C-terminus, but also cyclised forms in which the C-terminus of the distal ubiquitin was isopeptide-linked to Lys48 of the proximal ubiquitin [61]. This partitioning in to acyclic and cyclic diubiquitin was confirmed from mass values (differing by 18Da) and native MS interaction studies with the Znf_UBP domain of isopeptidase T, the UBD which recognises the free C-terminus of ubiquitin and which accordingly only formed complexes with the acyclic dimers [61, 62].

3.5 Use of isolated UBDs (C-terminal tail-directed) The exquisite binding selectivity of the Znf_UBP domain (of isopeptidase T) for the free C-terminus of ubiquitin (Figure 2) was also further exploited to selectively purify longer (than dimer) endogenous unanchored polyubiquitin chains [48]. Such polyubiquitin chains are increasingly being realised to play important roles in various aspects of cellular physiology [63]. This isopeptide linkage-independent UBD [62] was able to capture unanchored chains containing as many as 15 ubiquitins, including Lys48 and Lys11 linkages, from mammalian tissue and cell extracts [48]; further, the Znf_UBP domain was used to show that levels of unanchored chains increased when the 26S proteasome was pharmacologically inhibited [64]. The same strategy was also applied to the purification of endogenous unanchored polyubiquitin from plant and yeast extracts, made possible because of the high conservation of the ubiquitin sequence [64]. Placing the Znf_UBP domain back in its natural context of the full-length


(catalytically-inactive) USP5 protein along with its tandem UBA domains, generated a tool with a preference for longer unanchored polyubiquitin chains in pull-downs [64]. The Znf_UBP domain of USP5 was also used as part of the PSAQ approach to quantify cellular ubiquitin pools [19]; here, the UBD was used to capture and determine levels of both endogenous free (unconjugated) ubiquitin, including unanchored chains, as well as total ubiquitin after conversion of all ubiquitin conjugates to free monoubiquitin with a broad-spectrum DUB.

4. UBDs as cellular probes and sensors UBDs whether organised in artificial tandem arrays [12], as with TUBEs, or in isolation [65], have found application as in vivo biosensors of polyubiquitin. Here, UBDs with intrinsic selectivity for a defined polyubiquitin chain linkage, including Lys63-linked (utilising the UIMs of RAP80 and Vps27 or the TAB2 Npl4 zinc finger) or linear (UBAN) polyubiquitin, are fused to GFP. This generates a reagent which can selectively monitor the localisation and accumulation of the polyubiquitin chains in vivo. Such reagents have for example confirmed the role of Lys63-linked polyubiquitin chains in DNA damage repair following DNA double strand breaks (DSB), and in mitochondria-specific autophagy (mitophagy) [65].

However a caveat related to potential masking and/or stabilisation of polyubiquitin signals by the GFPtagged fusions and the associated continued fluorescent signal, has led to the development of an alternative approach in which biomolecular-induced fluorescence complementation is employed for the specific detection of Lys48-linked polyubiquitin chains in vivo [66]. An overview of this assay termed ubiquitination-induced fluorescence complementation (UiFC) is highlighted in Figure 6. Briefly two tandem arrays of three Epsin UIMs are fused to terminal fragments of the Venus fluorescent protein, with the concurrent binding of both tandem arrays to the Lys48-linked polyubiquitin chain bringing the two Venus fragments into close proximity, reconstituting fluorescence. This technique was used to highlight a role for Lys48-linked chains in the ER-associated degradation (ERAD) pathway. It is anticipated that this repertoire of tools will be expanded as further linkage selective domains are revealed; consequently such tools pave the way for the generation of an array of reagents to selectively


monitor polyubiquitin chains of defined linkage in vivo, offering insight into ubiquitin signalling in its endogenous setting.

5. UBDs as inhibitors The polyubiquitin sensors discussed in section 4, not only allow ubiquitin signals to be ‘tracked’ or visualised in living cells, but also result in the transient inhibition of pathways which rely on the specific chains targeted. This was evidenced from read-outs of TNF and IL-1-mediated NF-B signaling following expression of NZF- and UBAN-based sensors in cell models [65]. For example expression of the UBAN-GFP sensor, which targets linear polyubiquitin chains, was associated with inhibition of nuclear accumulation of p65 (RelA) after 15 minutes of TNF-stimulation, whilst untagged tandem fusions of the same domain have also been found to suppress LUBAC-induced NF-κB activation in reporter assays [67]. Conversely NZF-based sensors, which target Lys63-linked polyubiquitin chains, did not affect p65 accumulation consistent with the fact that these chains are not essential for efficient TNF-mediated NF-B signalling. Thus sensors can also be used to selectively inhibit ubiquitindependent signalling processes in cells.

Indeed as noted above an unexpected property of TUBEs developed for the affinity purification of ubiquitin-modified proteins was their ability to also act as inhibitors of DUBs and of proteasomal degradation of ubiquitin-modified substrates, presumably because their avid binding to the ubiquitin modifications competitively inhibits recognition by UBDs found at either the proteasome or within DUBs, which themselves use molecular recognition mechanisms based on UBDs. This mimics effects reported for ubiquitin-binding proteins, which in some cases have the ability to sequester ubiquitinmodified binding partners protecting them from deconjugation or degradation [68-70].

UBDs can also afford protection of the ‘parent’ proteins in which they reside. An interesting example is Met4 (in yeast), which contains a UIM and UIM-like domain, which act in tandem to limit the length of Lys48-linked polyubiquitin modification and consequently interaction with the proteasome, stabilising Met4. However under conditions of nutrient stress, the tandem UBDs of Met4 are inactivated, leading


to rapid proteasomal degradation [71]. This feature is found to be ‘portable’, with fusions of these domains stabilising other ubiquitinated substrates e.g. Sic1 by preventing their proteasomal degaradation [71], and could be exploited in a research context to experimentally manipulate protein half-lives. A similar property of UBA domains, as transferable stabilisation signals, was previously noted [72]. In the same way that exogenous UBDs (e.g. in TUBEs) can impact on recognition of ubiquitinmodified proteins by the proteasome, endogenous free S5a in cell is speculated to act as a chaperone, inhibiting the formation of forked polyubiquitin chains, thus maintaining efficient degradation at the proteasome [73]. Indeed this property is retained by S5a and its isolated UBDs in vitro; formation of forked polyubiquitin chains, formed in in vitro ubiquitin conjugation reactions by the E2 UbcH5 in concert with ring-finger and U-box E3s, can be prevented by the inclusion of S5a protein or GST-UIM sequences [74]. This property may find utility in directing the in vitro production of polyubiquitin chains of ‘standard’ isopeptide linkage for research purposes.

Isolated UBDs have found further applications as selective inhibitors of various (patho)physiological processes including preventing the aggregation of the polyglutamine protein huntingtin, the expansion of which is associated with the aetiology of Huntington’s disease [75]. The expanded mutant huntingtin proteins form insoluble aggregates that are associated with components of the ubiquitin system, including ubiquitin-binding proteins. In this case expression of GST-tagged UIM sequences from ataxin-3 and S5a were specifically found to inhibit the aggregation of mutant huntingtin in a cellular model, suggesting that a similar strategy might be considered to therapeutically target huntingtininduced toxicity in vivo. Further, expression of the Lys63-polyubiquitin-selective UBZ domain of the E3 enzyme RAD18 was able to inhibit the ubiquitin-dependent recruitment of DNA damage response factors to DNA repair foci following DNA DSB [76].


6. Other applications of UBDs UBDs have been used in a variety of other ways to manipulate ubiquitin-mediated processes. For example a strategy, termed Ubiquitin Ligase Substrate Trapping, has been developed, for the identification of substrates of E3 ligase enzymes [13]. Here, UBA domain sequences (from Rad23 or Dsk2) are artificially fused to different F-box proteins (SCF components), and bind the nascent ubiquitin chain of the modified ligase substrate(s); complexes are enriched via imuunoprecipitation targeting a FLAG epitope incorporated in the fusion protein which are the subject to MS-based identification. This strategy was able to identify known and novel substrates of eight different F-box proteins [13]. E3 ligase function was also probed in a high-throughput screening-compliant assay making use of Rad23-UBA2 on a coated 96-well plate, used to capture ubiquitinated proteins generated by E3 activity [77]. Here the captured modified proteins were then quantified by antibody-based methods. An extension of this assay made use of biotinylated TUBE1 to detect a polyubiquitin modification on auto-ubiquitinated GST-tagged E3s (autoubiquitination being a measure of E3 function) [78]. The biotinylated (ubiquitin-bound) TUBE is complexed with Streptavidin-Acceptor, and E3 is probed with a donor-labelled anti-GST antibody, bringing the acceptor fluorophore within close proximity of the donor-labelled antibody, generating an excitation-induced signal. This allowed the degree of autoubiquitination of the E3s MuRF1 and TRIM25 to be quantified. A related version of the assay used GST-tagged TUBEs (1 and 2), and a protein A acceptor-bead linked to E3-specific antibody, to quantitate Mdm2 activity [78].

In the context of E2 activity, fusion of the high affinity Znf_UBP domain of isopeptidase T to the E2 enzyme UBE2s, replacing its lysine-rich tail that is normally targeted by cis-autoubiquitination, generated an enzyme capable of producing significantly increased levels of (free) polyubiquitin chains including those which are Lys11-linked [25]. This refinement was central to the production of sufficient levels of chains of this linkage, permitting the first structural studies.


7. Summary and future perspectives In summary we have highlighted the diverse range of applications of UBDs as tools with respect to the enrichment, isolation and identification/detection of ubiquitin-modified proteins (see Figure 6), and some of the biological insights gained from these uses of UBDs (Table II). Future challenges include the need to develop a portfolio or toolbox of UBD-based reagents to allow all polyubiquitin linkage types to be probed. Such reagents may already exist in nature but have been overlooked due to the (until recently) limited availability of polyubiquitin chains of defined linkages required for the identification and characterisation of the UBDs. Alternatively, nature may have solved the question of recognition of polyubiquitin chains of certain linkages by alternative strategies. These could include the use of UBDs donated by different ubiquitin-binding proteins in the same multi-protein complexes (similar to the UBAN coiled-coil), or indeed selectivity may be achieved in vivo by simple compartmentalisation of ubiquitin-binding protein and its particular target. If such complex mechanisms are found to be a generality, innovative solutions involving engineered, synthetic protein sequences for example using phage display approaches [79] may be required to generate synthetic reagents with polyubiquitin linkage selectivity for certain linkage types. Tools to distinguish unanchored from conjugated polyubiquitin are certainly feasible [48] given the existence of UBDs that selectively target ubiquitin’s exposed C-terminus [33]. Polyubiquitin chain length-selective reagents (i.e. molecular rulers) would be of particular importance and these might also be developed with inspiration from recognition mechanisms already highlighted in nature [39]. Likewise, tools that permit monoubiquitinated proteins to be selectively isolated over polyubiquitin chains would have obvious utility. Given the added complexity of polyubiquitin modifications revealed from the characterisation of mixed linkage and branched polyubiquitin chains [3, 23], it is difficult to envisage how these types of polyubiquitin modifications (certainly the latter) might be specifically targeted. Finally one might envisage, in the future, bidentate reagents with the ability to simultaneously recognise protein substrates with defined (poly)ubiquitin modifications. Once more nature already reveals such recognition events; as an example, certain TLS polymerases contain UBDs in tandem with other domains that mediate specific target (e.g. PCNA) interactions [80]. Artificial sequences with the same properties – the ability to recognise both target protein and its PTM - would potentially offer the opportunity to enrich/purify


biomedically-relevant proteins modified with polyubiquitin chains of defined linkage (that may be required for optimal bioactivity), the possibility to protect/inhibit the disassembly of polyubiquitin chains on a single protein, and to facilitate the monitoring of dynamic changes in ubiquitin modifications of individual proteins in vivo. Acknowledgements Current work in the author’s laboratories is funded by the BBSRC (ref. BB/I011420/1).

Conflicts of interest The authors declare no financial/commercial conflicts of interest.


Figure 1. (A) Structure of ubiquitin (pdb 1ubq) showing the position of Met1 and its seven lysine residues (pink, indicated K) used in isopeptide bond formation within polyubiquitin chains. Conjugation to substrate proteins (and neighbouring ubiquitins within chains) occurs via the Gly76 residue in ubiquitin’s exposed C-terminus. (B) Seven types of homogenous isopeptide-linked polyubiquitin chains (as well as linear Met1-linked peptide chains) are possible, with Lys11, Lys48 and Lys63 illustrated. Mixed linkage polyubiquitin chains have also been reported (e.g. involving different single lysine residues within the chain), as well as branched chains (where a single ubiquitin is modified at two or more lysines). (C) Covalent ubiquitination of selected targets (demoted x) with ubiquitin (black ‘tadpoles’), catalysed by a series of enzymes termed E1-E3, results in proteins which are singly or multiply monoubiquitinated, or modified with polyubiquitin chains of different topologies. Ubiquitin is activated by an E1 ubiquitin-activating enzyme, prior to transfer to the E2 ubiquitin-conjugating enzyme as a thiolester intermediate. An E3 ubiquitin ligase confers substrate recognition by selective substrate binding and mediates transfer of ubiquitin from the E2 to the selected substrate. The ubiquitin code is ‘read’ by more than 20 different families of ubiquitin-binding domains (UBDs) within intracellular ubiquitin-binding proteins (UBP). PTM with ubiquitin can be reversed by the actions of a UBP with deubiquitinating enzyme (DUB) activity.

A

K29 K33

B

K27

M1 K63 C-terminus

K11

K6

K48 homogenous

C

E1

E3

E1

E1

E2

(ubiquitin-activating enzyme)

X mixed linkage

E2 (ubiquitin-conjugating enzyme)

E1

E2

DUB

E3 (ubiquitin ligase; substrate selection)

X

E3

branched

E3 E2 X

X

X UBD UBP


Figure 1

Figure 2. (A) Different surfaces on ubiquitin are targeted by the non-covalent interactions of UBDs, commonly an Ile44 (I44 - red) centred hydrophobic patch, an acidic hydrophilic Asp58 (D58 - green) centred patch, and residues (G76 – blue) at the C-terminus of ubiquitin (when not conjugated to substrates). Representative UBDs which recognise these surfaces are indicated. (B) Structural model of a single ubiquitin in complex with three different UBDs (parent proteins indicated in brackets), generated from the overlay of the experimentally determined binary complexes, demonstrating that simultaneous interactions at different sites on ubiquitin are not mutually exclusive (model derived from pdb codes 2L00, 1WR1 and 2G45).

A

B

A20 ZnF (ZNF216)

I44

UBA (ubiquilin-1)

D58 A20 ZnF

D58

ubiquitin

ubiquitin I44

G76 UBA

G76 Znf_UBP Znf_UBP (IsoT/USP5)

Figure 2


Figure 3. (A) X-ray structure of the A20 ZnF4 domain (three copies) of the A20 protein (pdb 3oj3), showing simultaneous complexation with a single ubiquitin molecule via the Ile44 hydrophobic face (red), the TEK box motif using Thr9/Lys11 (pink) and the polar face centred on Asp58 (green); the A20 ZnF4 in each case also uses a different binding face (see also Figure 5B). (B) Structure of the ubiquitin complex of the nucleotide exchange factor Rabex5 (pdb 2c7n) with simultaneous interaction of a single ubiquitin with the MIU and A20 ZnF motifs of different Rabex5 molecules, again using the Ile44 (red) and Asp58 (green) patches on ubiquitin.

A

B

UBD3

UBD2

T9

K11 ubiquitin

MIU

I44 I44

D58

ubiquitin D58 A20 ZnF

UBD1

A20 ZnF4 (A20) - ubiquitin

Rabex5 - ubiquitin

Figure 3


Figure 4. Linkage-dependent conformational selection and avidity effects in polyubiquitin recognition by UBDs. (A) When different lysine residues are used within isopeptide-linked ubiquitin dimers, the chains adopt different topologies that present different recognition surfaces. Lys63-linked dimers exhibit an open extended structure where the UBD interaction surfaces (e.g. Ile44, red) are exposed in both the proximal (green) and distal ubiquitin molecules (blue grey). In contrast, in Lys48-linked dimers the Ile44 binding surfaces of both distal and proximal ubiquitin motifs form weak contacts resulting in an equilibrium between open/extended and closed/compact forms. More precisely, different polyubiquitin chains present their UBD interaction surfaces within a defined region of conformational space. Individual UBD-containing proteins and complexes select conformations sampled by these interaction surfaces that are unique to different chains topologies. Blue arrow denotes free C-terminus of the proximal ubiquitin which is shown in green (and depicts the proximal ubiquitin of both Lys63- and Lys48-linked dimer structures). (B) Multiple UBDs placed in tandem within the same primary sequence connected both by rigid or flexible linkers can interact simultaneously with different ubiquitin moieties leading to avidity effects that enhance binding affinity and contribute to polyubiquitin chain linkage selectivity. Here Rap80 interacts with Lys63-Ub2 using two rigidly connected UIMs in tandem within a single extended helix. The orientation and spacing of the two UIMs allows for selectivity by exploiting differences in the separation of the Ile44 binding sites for distal and proximal ubiquitin motifs in different extended diubiquitins, which favours the spacing within the Lys63-linked dimer (pdb 3A1Q). (C) In contrast, some UBDs simultaneously use two binding surfaces on the same UBD to interact with different ubiquitin moieties within a polyubiquitin chain. Here, UBA2 of hHR23A interacts with Lys48-Ub2 by selecting for binding to a more compact conformation (pdb 1zo6).


A

B

C

tandem UIMs (Rap80) – Lys63-Ub2

UBA2 (hHR23A) – Lys48-Ub2 Figure 4


Figure 5. Structural models rationalise polyubiquitin-selective recognition by selected UBDs. UBDs in complex with polyubiquitins are highlighted; blue spheres represent zinc ions and Ile44 of ubiquitin is indicated in red. (A) S5a/RPN10 achieves polyubiquitin selectivity via two flexibly linked helical UIMs that bind to the Ile44 patches on ubiquitin motifs (in this example within Lys48-linked diubiquitin) (pdb 2kde). (B) The A20 ZnF4 domain of the A20 protein is able to make multiple contacts with three different ubiquitins within a Lys63-Ub3 chain, via three individually weak ubiquitin-binding sites. One of the binding sites (an inverted UIM) recognises the classical ubiquitin Ile44 ubiquitin face (top right), with the two other patches using non-classical ubiquitin-binding sites involving the TEK box motif (Thr9-Lys11) and a loop region of ubiquitin (around residues Glu51-Lys63) (pdb 3oj3). (C) TAB2 contains a NZF UBD with two ubiquitin-binding faces that leads to weak linkage selectivity for Lys63-diubiquitin (shown) (pdb 3A9J). (D) The X-ray structure of the UBAN motif of NEMO (pdb 2ZVO) reveals that the distal and proximal ubiquitin motifs of a linear ubiquitin dimer are recognised by UIMs on different helices of a UBAN coiled-coil dimer. The distal (slate blue) ubiquitin is recognised mainly through the canonical Ile44 patch, but the proximal ubiquitin motif (green) forms specific interactions via Gln2 and Glu16 at its N-terminus; Arg71, Leu72 and Arg73 within the otherwise flexible preceding C-terminus of the distal ubiquitin are also targeted, demonstrating specific recognition of the linker region. The complex demonstrates selectivity by not using the Ile44 patch on the proximal ubiquitin, but rather residues (Phe4 or Gln2/Glu16) on the N-terminal -hairpin of ubiquitin close to the Met1 conjugation site. (E) The HOIL-1L subunit of the LUBAC also contains a NZF domain with the classical zinccoordinating core region and a second binding site within an extended helical tail region which together mediate interactions with Met1-linked linear diubiquitin. The NZF core recognises the Ile44 hydrophobic patch of the distal ubiquitin (left) while simultaneously binding to a Phe4-centred surface on the proximal ubiquitin (right, green) via the extended NZF helical tail (pdb 3B08).


Figure 5


Figure 6. Overview of the different applications of UBDs as tools to investigate the ubiquitin-modified proteome. (1) Artificial Tandem Ubiquitin-Binding Entities (TUBEs) can be used to directly purify (poly)ubiquitin-modified proteins (denoted x, black ‘tadpoles’ represent ubiquitin) and also act to inhibit DUB activity (and proteasomal degradation) in cell extracts. (2) Individual UBDs, or natural tandem UBD repeats, with specificity for different ubiquitin interaction surfaces, can be used to purify polyubiquitin and ubiquitin-modified proteins. Free (unanchored) polyubiquitin can be captured by UBDs with specificity for ubiquitin’s free C-terminus (purple). In some cases isolated UBDs retain binding specificity on beads; in other cases artificial avidity effects may mask intrinsic specificity. (3) UBDs form part of the Ubiquitin Ligase Substrate Trapping approach, where they are artificially fused to different F-box proteins (SCF components) and bind the nascent ubiquitin chain of the modified ligase substrate(s) which are then subject to MS-based identification. (4) Fusing linkage-selective individual and tandem UBDs to fluorescent tags allows ubiquitin-signalling events to be monitored in living cells. Ubiquitination-induced fluorescence complementation (UiFC) involves two tandem arrays of three Epsin UIMs fused to terminal fragments of the Venus fluorescent protein. Simultaneous binding of both arrays to Lys48-linked polyubiquitin chains brings the two Venus fragments into close proximity reconstituting fluorescence. (5) Expression of individual UBDs in mammalian cells can be used to competitively inhibit selected ubiquitin-mediated processes.

Figure 6





1

Table I – Selected UBDs and their properties Ubiquitin binding domain (UBD)

Representative e.g.

Ubiquitin surface

Ubiquitin selectivity

Applications

Notes

S5a/RPN10

(UIM-1 and UIM-2) I44 (UIM-2 only) L8, V70, L71

Longer polyubiquitin chains (relative to monoUb), both Lys48- and Lys63-linked. Other linkages not determined (N.D.).

Polyubiquitin purification, competitive inhibitors

Full length S5a and isolated UIMs have been used for polyubiquitin purification. S5a/GST-UIM in vitro prevents formation of forked polyubiquitin chains. In a cellular model, S5a-UIM expression inhibits aggregate formation of mutant huntingtin.

Rap80

I44

Lys63-, relative to Lys48-linked polyubiquitin chains. Increased affinity for longer chains. No binding to linear polyubiquitin chains.

Polyubiquitin purification, TUBES

GFP-fused (natural) RAP80 UIMs, and artifically enhanced tandem UIMs (with a structured linker between UIMs) have been used as polyubiquitin linkage specific sensors.

hHR23A UBA1

I44

No reported selectivity.


TUBEs have been generated based on hHR23A UBA1, and bind Lys48- and Lys63-linked polyubiquitin chains with high affinity relative to the UBD alone.

Ubiquilin-1

I44



TUBEs have been generated based on ubiquilin-1 UBA, and bind Lys48- and Lys63-linked polyubiquitin chains with high affinity relative to the UBD alone.

UBAN

ABIN

I44 and Phe4 or Gln2/Glu16

Linear, relative to Lys48- and Lys63-linked polyubiquitin chains.

Polyubiquitin sensors

A GFP-fusion with the UBAN domain or (three) tandem UBAN domains has been used as polyubiquitin linkage specific sensors.

Npl4 zinc finger

NZF

TAB2

I44

Lys63-, relative to Lys48-linked and linear polyubiquitin chains.

Polyubiquitin sensors

A GFP-fusion with TAB2 NZF has been used as a polyubiquitin linkage specific sensor.

A20 zinc finger

ZnF A20

ZNF216

D58


Polyubiquitin purification

The Znf_A20 domain has been used to capture polyubiquitinated substrates and unanchored polyubiquitin species (including dimers).

Ubiquitin-binding zinc finger

UBZ

RAD18

N.D.

Lys63-linked polyubiquitin, relative to Lys48-linked polyubiquitin chains.

Competitive inhibitor

Exogenous expression of RAD18's UBZ domain in cells inhibits recruitment of ubiquitin-dependent proteins to DNA repair foci.

Zinc finger ubiquitin binding domain

Znf UBP (BUZ)

isoT/USP5

C-terminal GG


Polyubiquitin purification

Affinity for free C-terminus permits use of Znf_UBP domain for purification of unanchored polyubiquitin chains.

UIM

-family

Ubiquitininteracting motif

Ubiquitinassociated domain

ZnF family

Ubiquitin binding in ABIN and NEMO

1

UBA

Examples of some of the more than 20 UBDs (only ZnF and -helical families shown) relevant to this review. For a more comprehensive list see reference [4].

This article is protected by copyright. All rights reserved.




Table II - Applications of TUBEs in the purification of ubiquitin-modified proteins Application

System

Reagent composition

Function of TUBE To monitor the ubiquitinated substrates which accumulate following DUB inhibition

Monitoring the activity of DUBs

Mammalian

a

TUBE 1 and 2

TUBE 1

To assess the interplay between the DUB UCHL1 and the high affinity choline transporter To identify portable degrons which are ubiquitinated, which can serve as tools to monitor proteasome activity To capture monoubiquitylated proteins, in this case IB

References [81] [82] [83]

[84]

[85]

[86]

TUBE 2 To assess ubiquitination of KCa3.1, a membrane protein, during endocytic trafficking and lysosomal degradation To assess the oligomerisation and ubiquitination of p53 following the introduction of point mutations

Mammalian Monitor the ubiquitination of proteins of interest

To assess the ubiquitination of pro-IL-1β

[87]

[88]

[89]

TUBE 1 and 2 To compare the ubiquitination of wild-type and a disease associated mutant PABPN1 Monitor ubiquitinated proteins involved in DNA damage response

Yeast Effect of protein of interest on autophagic clearance

Mammalian

TUBE 1

TUBE 2

TUBE 1 Identification of novel ubiquitinated proteins

Mammalian TUBE 2

This article is protected by copyright. All rights reserved.

Monitor the ubiquitination of histones Assess the role of TBK-1 in the regulation of the autophagic degradative pathway of numerous cargo Identify novel targets of ubiquitination in the NOD2mediated inflammatory signalling Used to identify and purify ubiquitinated proteins involved in the induction of osteoarthritis

[90]

[91]

[92]

[93]

[94]

[95]

TUBE 1 and 2

Plant

To identify and monitor ubiquitylation machinery

Non-selective capture of ubiquitinated substrates Purification of heterogeneous chains of ubiquitin/ubiquitinlike proteins a

Arrangements based on the UBA domains of hHR23A

Yeast

5 repeats of Dsk2-UBA

Fungus

TUBE 2

Mammalian

TUBE 2

Mammalian

TUBE 1 and 2

Mammalian

TUBE 2

Consider ubiquitination pattern following cytokine induction of nitric oxide synthase To study global ubiquitylation events that may lead to the identification of potential drug targets Analysis of the Arabidopsis ubiquitome To purify ubiquitinated proteins following DNA damage e.g. Def1, RNA polymerase II Identify targets of ubiquitination in nitrogen stress conditions in Magnaporthe oryzae As part of a multidimensional approach to identify substrates of E3 ubiquitin ligases To consider the activity of the E3 ubiquitin ligase muscle atrophy F-box in the ubiquitination of IkB As a control for binding nonselectively to ubiquitinated substrates

Revealed heterologous SUMO-2/3 ubiquitin chains after TNF stimulation

TUBE 1 represents four repeats of UQ1-UBA, TUBE 2 represents four repeats of hHR23A–UBA1


[96]

[97]

[98]

[99]

[100]

[101]

[102]

[23] [103]

[104]

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