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The UBE2E as Conjugating Dispersers: Extending Function with Extended Extensions

Vladimir N. Uversky

Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

Correspondence to Vladimir N. Uversky: Department of Molecular Medicine, University of South Florida, College of Medicine, 12901 Bruce B. Downs Boulevard, MDC07, Tampa, FL 33612, USA. [email protected] http://dx.doi.org/10.1016/j.jmb.2013.07.024 Edited by S. Khorasanizadeh

In this issue of the Journal of Molecular Biology, as ID proteins (IDPs) and hybrid proteins possessing Schumacher et al. investigate the molecular mech- ordered domains and IDP regions (IDPRs) [10].IDPs/ anisms underlying the ability of the members of IDPRs are very common in nature [11], possess wide Class II -conjugating enzymes (E2s) to structural heterogeneity [12], and are abundantly catalyze the formation of polyubiquitin chains [1].In involved in various biological processes, where they addition to the highly conserved catalytic domain play crucial roles by regulating functions of their carriers (the core ubiquitin-conjugating domain, UBC, of (IDPRs) or binding partners (IDPs and IDPRs) [3–9]. ~150 residues), the human UBE2E proteins studied Importantly, intrinsic disorder is unevenly distributed here contain N-terminal intrinsically disordered (ID) within the hybrid proteins, being typically more extensions of different lengths [2]. The authors common at termini [13]. Many biological suggest that these extended extensions are a key functions are unique for disordered termini and cannot for extending the E2 functionality. The impact of this be found in other disordered parts of a protein, important observation goes far beyond the under- suggesting that these disordered tails are not simple standing of how the ubiquitination machinery acts, flexible protrusions but are evolved to serve [13]. since (a) it argues that studying the intact proteins, Ubiquitination is an intriguing and unique post- not just their core catalytic domains, is important for translational modification (PTM) of proteins. This complete understanding of protein functionality, and intriguing nature and uniqueness of ubiquitination are (b) it provides new clues on how the protein obvious at several levels. First, this PTM represents a functionality can be controlled and modulated via covalent attachment of ubiquitin or one of the ubiquitin- non-catalytic and therefore seemingly useless like proteins [Ubls, such as small ubiquitin-like modifier extended extensions, ID regions. A brief descrip- (SUMO), interferon-induced 15-kDa protein (ISG15), tion of what is extended (i.e., a short description of or neural precursor cell expressed developmentally the protein intrinsic disorder phenomenon with the down-regulated protein 8 (NEDD8)] [14] to a target focus on disordered tails) from what (i.e., an protein, whereas the vast majority of other PTMs are overview of the ubiquitination machinery with the based on the covalent attachments of relatively small focus on UBE2E proteins) is given below to help chemical groups. Therefore, the ubiquitin or Ubl protein better appreciate the importance of these attachment provides a large interaction surface by conclusions. which the modification can be decoded [15,16]. The last decade and a half witnessed an increased Second, ubiquitination is known to be delivered in recognition that a biologically active protein is not several flavors, and one target protein can be mono-, obligated to possess a unique three-dimensional multi-, and polyubiquitinated, depending on the structure as a whole or in part and that many number and mutual configuration of attached ubiqui- biologically important functions may originate from tin molecules [17–19]. Here, the multiubiquitination the lack of ordered structure in a protein molecule [3– is a result of multiple lysine residues of one target 6], and these functions are complementary to the protein being used for the ubiquitin attachment functions of ordered proteins [3,7–9]. Such structure- [17,19], whereas polyubiquitination can be produced less functional proteins and protein domains are known when the lysines of the substrate-conjugated

0022-2836 © 2013 The Author. Published by Elsevier Ltd. Open access under CC BY license. J. Mol. Biol. (2013) 425, 4067–4070 4068 The UBE2E Proteins as Conjugating Dispersers ubiquitin(s) are used as subsequent ubiquitin attach- ligase is required for assembly of a polyubiquitin ment points [18,19]. The complexity of this PTM and chain on a protein acceptor [16]. its outputs is further increased by the fact that E2s are grouped into four structural classes polyubiquitin chains can be “linear” (no more than depending on the presence/absence and the local- one amino group of each ubiquitin is linked to ization of the non-catalytic extensions relative to the another ubiquitin) or “branched/forked” (at least one UBC domain [2]. Class I includes UBC-only E2s, ubiquitin is attached to other via two or classes II and III consist of E2s possessing the more different amino groups) [16,20]. Also, the catalytic domain and additional N- or C-terminal polyubiquitin chains can also be mixed and contain extensions, respectively, whereas E2s having both other heterologous proteins from the ubiquitin family N- or C-terminal extensions belong to class IV. In such as SUMO [16,21]. human ubiquitinome, there are at least 14, 9, 9, and Third, due to the large interaction surface and 3 E2s in classes I, II, II, and IV, respectively [2]. added complexity due to the ability to be delivered in Earlier bioinformatics and computational analyses mono-, multi-, and polyubiquitinated forms, this PTM revealed that the C-terminal acidic domains of the is not only crucial for the control of almost all cellular class III E2s possess all the features typical of the processes by mediating the regulated degradation of extended IDPs/IDPRs, with high mean net charge proteins, but it often defines the fate of a modified and low mean hydropathy being the most obvious protein affecting transcriptional regulation, traffick- characteristics [25]. Furthermore, this study empha- ing, endocytosis, lysosomal targeting, and other sized that the acidic ID domain of the class III E2s, types of targeting/localization, protein–protein inter- which is involved in recognition of ubiquitin and/or E3 action, complex assembly, modulation of function or ligase, is functionally important [25]. stability, and so on [18]. Furthermore, multiubiquiti- The work by Schumacher et al. continues to shed nation and polyubiquitination can generate diverse light on the role of intrinsic disorder in the function of substrate–ubiquitin structures, defining different the ubiquitination machinery [1]. Using a set of fates of target proteins [22,23]. For example, DNA experimental and computational tools, the authors repair and expression are regulated by mono- showed that the human members of the class II E2s, ubiquitination, whereas K48 proteins are typically UBE2E1, UBE2E2, and UBE2E3, contain ID N-ter- targeted for proteasomal degradation, while K63 minal tails. This is illustrated in Fig. 1, which shows polyubiquitination is needed for regulation of kinase available crystal structures for human UBE2E1 and activation, DNA damage tolerance, signal transduc- UBE2E2 (Fig. 1a and b, respectively) and predicted tion, and endocytosis [22]. disorder propensities of UBE2E1, UBE2E2, and Fourth, in contrast to many other PTMs, where the UBE2E3 (Fig. 1c). Due to the high propensity for modifying group is added to the target protein by one intrinsic disorder, the N-terminal extension either is enzyme, ubiquitination (which is the attachment of mostly absent from the protein structure [as shown in ubiquitin or Ubl protein to lysine residues on a target Fig. 1a, where only 7 of 41 N-terminal residues are protein via the formation of the isopeptide bond detected as a short structured region (residues 21– between the C-terminus of ubiquitin and an amine 27)] or was deleted from a prior protein crystallization from the lysine of the substrate protein) requires a (as shown in Fig. 1b). Figure 1c shows that the UBC complex cascade of catalytic reactions that involves domains of UBE2E1, UBE2E2, and UBE2E3 are three classes of enzyme, an E1 ubiquitin-activating predicted to be ordered and conserved and also enzyme (also known as Uba1), an E2 ubiquitin-con- shows that the N-terminal extensions of these three jugating enzyme (also known as Ubc), and an E3 E2s are all predicted to be mostly disordered. [18]. Although this is an interesting observation, the Finally, fifth, the complexity of this PTM is further even more intriguing twist is how this disordered tail increased by the fact that the ubiquitinome (i.e., a set is used by UBE2Es. The authors show that the UBC of protein involved in the ubiquitination or the domains of E2s, being present alone, are responsi- ubiquitin pathway) is large and intriguingly inter- ble for the polyubiquitination of target proteins, twined. For example, contains whereas this polyubiquitinated capability is limited several Uba1 or E1 enzymes, more than 50 Ubcs when the UBC domains are decorated with the or E2s, and several hundred E3s [24]. E2s are disordered N-terminal tails, since the full-length recognized as key players of the ubiquitin pathway, UBE2E proteins primarily promote protein mono- since they control the fate of the target proteins by ubiquitylation. Therefore, the highly flexible nature of modulating processivity and topology of the poly- the disordered N-terminal extensions is used by ubiquitin chain assembly [25]. Furthermore, although UBE2Es to limit the processivity of their catalytic many E2s synthesize multiple types of linkages, an domains. In other words, the disordered tail prevents interplay between the various E2s and E3s not only polyubiquitin chain building by somehow restricting is crucial for targeting specific proteins for ubiquitina- the donor ubiquitin molecule from accessing the tion but also defines the length and linkage type of acceptor ubiquitin at the E2 active site. Although the polyubiquitin chains, and in most cases, the E3 nature of this interaction is uncertain, it is possible The UBE2E Proteins as Conjugating Dispersers 4069

AB21

27 42

UBE2E1 (1-193, 3BZH) UBE2E2 (54-201, 1Y6L) C 1.0

0.8

0.6

0.4 PONDR-FIT score PONDR-FIT

0.2 UBE2E1 UBE2E2 UBE2E3

0.0 0 50 100 150 200 Residue number

Fig. 1. Order and disorder in human UBE2E proteins. (a) Crystal structure of human UBE2E1 [Protein Data Bank (PDB) ID: 3BZH]. Although the full-length protein was used in the crystallization experiments, the N-terminal domain (residues 1–41) is mostly unresolved. In fact, residues 1–20 and 28–41 are expected to be disordered since they are located in regions of missing electron density. (b) Crystal structure of human UBE2E2 (PDB ID: 1Y6L). N-terminally truncated protein with removed N-terminal residues 1–53 was used crystallized. (c) PONDR-FIT plot representing disorder propensities in human UBE2E1 (red continuous line and pink shade), UBE2E2 (blue broken line and cyan shade), and UBE2E3 (orange dot/dash line and yellow shade). 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