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Although N-terminally ubiquitinated proteins were identified in cells many years ago, an enzyme with this specific

activity, Ube2w, has only recently been reported. Despite characterization of its activity, the source of its specificity for particular N termini and for the ability to distinguish the N terminus from lysine side chains remained unclear. In this issue, Vittal et al.1 report on the structure of Ube2w, which contains noncanonical structural characteristics, favoring the recognition of disordered N termini (Fig. 1a) rather than primary sequence motifs or structured epitopes.

Ubiquitination, the process of attaching the small modifier protein ubiquitin to other proteins, controls the fate of thousands of proteins by regulating protein activity, complex formation, protein localization and transcription2. Ubiquitination involves the coordinated action of a cascade of three enzymes, a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3), to attach ubiquitin to lysine residues in substrates, more specifically to the ε-amino group of lysine side chains2. However, other attachment sites have also been reported, such as the α-amino N termini of substrates. The proteins MyoD, the E7 oncoprotein from human papillomavirus type 16, latent membrane protein 1 from Epstein-Barr virus and inhibitor of differentiation 2 have been reported to be N-terminally ubiquitinated by Ube2w, the only E2 shown to ubiquitinate substrates at their N termini3,4. One of the distinguishing characteristics of Ube2w compared to other E2 enzymes is the presence of an atypical catalytic center, in which a histidine residue replaces an asparagine that is highly conserved among most E2s. This unique catalytic center has been demonstrated to aid in selecting α-amino termini rather than ε-amino groups on lysine side chains in Ube2w-mediated ubiquitination4. But one remaining question is how Ube2w selects its specific substrates.

Vittal et al.1 used in vitro ubiquitination assays to demonstrate that the backbone of N-terminally disordered substrates contacts Ube2w; the enzyme does not recognize

three-dimensional epitopes, secondary structural elements or primary sequence motifs (Fig. 1a–d). Thus, the sequence of potential substrates is only relevant in the sense that certain sequences promote disorder as opposed to a compact folded structure. Using hemagglutinin (HA)-tagged ubiquitin in their assays, Vittal et al.1 observed the formation of a linear polyubiquitin chain on the N terminus of their substrate instead of the addition of a monoubiquitin moiety (Fig. 1e). In this case, the HA tag acted as a disordered N terminus and thus as a substrate itself.

Ube2w was previously known to form weak dimers, which are not important for function as a monomeric mutant was

similarly catalytically active5. A recent crystal structure of Ube2w lacking the C terminus showed a domain-swapped dimer but provided limited functional insight6. To determine a structural ensemble of full-length Ube2w, Vittal et al.1 used NMR spectroscopy and the ROSETTA algorithm, which allowed visualization of the noncanonical features of Ube2w responsible for its activity toward α-amino N termini. The structural ensemble depicted the C-terminal region of Ube2w as flexible, adopting multiple conformations relative to the catalytic core, potentially providing a platform for recognizing many and varied substrates1.

What is the function of N-terminal ubiquitination? Although it may

ProtEin DiSorDEr

Wagging a tail at ubiquitinThe ubiquitin-conjugating enzyme Ube2w monoubiquitinates proteins with disordered N termini and may target lysine-less proteins for degradation.

tanja mittag & melissa r marzahn

Figure 1 | Ube2w monoubiquitinates the disordered N termini of proteins. (a–d) Ube2w attaches one ubiquitin (orange) to proteins with disordered N termini (a) but not with disordered C termini (b), disordered loops (c) or structured (for example, α-helical) N termini (d). (e) Ube2w can build long polyubiquitin chains with N-terminally HA-tagged ubiquitin (red) because the HA tag is intrinsically disordered and serves as a substrate itself. Disordered regions are indicated as dashed lines. (f) In many cancers, a substantial fraction of cases have alterations in Ube2w, mostly amplifications; here, the frequency of alterations is plotted against data from individual cancer genomics studies. Overall, 54% of all cancer sequencing studies deposited in http://www.cbioportal.org/ (ref. 10) have reported cases with altered Ube2w. Data sets for published studies were compiled from the literature. Data sets for provisional studies at The Cancer Genome Atlas (TCGA) stem from Broad Firehose. MSKCC, Memorial Sloan-Kettering Cancer Center; MICH, University of Michigan, 2012; chRCC, chromophobe renal cell carcinoma; ccRCC, clear cell renal cell carcinoma; ACC, adenoid cystic carcinoma; AML, acute myeloid leukemia.

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8 nature chemical biology | VOL 11 | JANUARY 2015 | www.nature.com/naturechemicalbiology

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ultimately target proteins for degradation by the proteasome, N-terminal monoubiquitination most likely serves to prime subsequent polyubiquitination mediated by additional enzymes3,7. The functional reasons for this complicated chain of events are unclear at this point, although one possibility is that N-terminal ubiquitination clears nascent lysine-less polypeptide chains from stalled ribosomes in concert with the ribosome quality control complex. Additionally, as 60–90% of all proteins in the cell are N-terminally acetylated, cotranslational N-terminal ubiquitination could represent a competing mechanism for regulation1.

In the future, defining the N-terminal degron more specifically will be crucial for an improved understanding of substrate specificity. Several open questions remain: (i) What is the shortest disordered sequence that supports recognition by Ube2w? (ii) How is recognition influenced by sequence-disorder relationships such

as net charge and charge patterning? These properties strongly influence conformational sampling, compactness of the disordered region and accessibility for binding partners8. (iii) How does monoubiquitination of a disordered N terminus influence proteasomal degradation, as degradation is thought to require a long disordered tail or internal loop9? (iv) Does N-terminal ubiquitination interfere or cooperate with substrate recognition by the proteasome?

Although it will require technical innovation to specifically enrich N-terminally ubiquitinated proteins, large-scale analysis of this modification within the proteome will provide insights into the pervasiveness of this new type of ubiquitin-related regulation. Notably, Ube2w is amplified in a substantial fraction of prostate, liver, breast and stomach cancers (http://www.cbioportal.org/)10, suggesting that its overactivation may lead to aberrant degradation of important regulatory

proteins, highlighting an important role in maintaining homeostasis (Fig. 1f). ■

Tanja Mittag and Melissa R. Marzahn are at the Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. e-mail: tanja.mittag@stjude.org

references1. Vittal, V.S. Nat. Chem. Biol. 10, 83–89 (2015).2. Streich, F.C. Jr. & Lima, C.D. Annu. Rev. Biophys. 43,

357–379 (2014).3. Tatham, M.H., Plechanovova, A., Jaffray, E.G., Salmen, H. &

Hay, R.T. Biochem. J. 453, 137–145 (2013).4. Scaglione, K.M. et al. J. Biol. Chem. 288, 18784–18788 (2013).5. Vittal, V., Wenzel, D.M., Brzovic, P.S. & Klevit, R.E.

Cell Biochem. Biophys. 67, 103–110 (2013).6. Sheng, Y. et al. Mol. Cell. Proteomics 11, 329–341 (2012).7. Christensen, D.E., Brzovic, P.S. & Klevit, R.E.

Nat. Struct. Mol. Biol. 14, 941–948 (2007).8. Babu, M.M., Kriwacki, R.W. & Pappu, R.V. Science 337,

1460–1461 (2012).9. Inobe, T., Fishbain, S., Prakash, S. & Matouschek, A.

Nat. Chem. Biol. 7, 161–167 (2011).10. Gao, J. et al. Sci. Signal. 6, pl1 (2013).

Competing financial interestsThe authors declare no competing financial interests.

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