Modes of allosteric regulation of the ubiquitination machinery

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Ubiquitination is a post-translational modification crucial for cellular signaling. A diverse range of enzymes constitute the machinery that mediates attachment of ubiquitin onto target proteins. This diversity allows the targeting of various proteins in a highly regulated fashion. Many of the enzymes have multiple domains or subunits that bind allosteric effectors and exhibit large conformational rearrangements to facilitate regulation. Here we consider recent examples of ubiquitin itself as an allosteric effector of RING and RBR E3 ligases, as well as advances in the understanding of allosteric regulatory elements within HECT E3 ligases.

Introduction

This review first covers the basics of ubiquitination, highlights the different modes of allosteric regulation of the ubiquitination machinery, and then examines specific examples. Ubiquitination involves the covalent attachment of ubiquitin, an 8.6 kDa protein, to target proteins. This post-translational modification (PTM) is found in eukaryotes and is involved in proteasome degradation and many other signaling pathways [1,2]. An enzymatic cascade mediates the formation of an isopeptide bond between the C-terminus of ubiquitin and the amino group of a lysine sidechain on the target protein. Ubiquitination involves three steps: 1) activation of ubiquitin C-terminus by an E1 enzyme in a fast, ATP-dependent reaction, 2) conjugation onto an E2 via transthiolation with ubiquitin-charged E1, and 3) ligation onto the target by an E3 which interacts with both ubiquitin-charged E2 and the target (Figure 1a). When the target is ubiquitin itself, which contains seven lysines (Figure 1b), ubiquitin chains are formed [2]. Removal of ubiquitin from the target is achieved via de-ubiquitinases (DUBs) making ubiquitination a reversible PTM [3,4]. In addition to lysine, the N-terminal amino group, and some other side chains can be ubiquitinated [5].

While there are only a few E1 enzymes encoded in the human genome, there are tens of E2s and DUBs, and hundreds of E3s [8,9]. This diversity likely reflects the need to ubiquitinate a range of target proteins in a highly regulated fashion. Three types of E3 ligases — RING/U-box, HECT, and RBR (vide infra) — have conserved catalytic architectures and utilize different mechanisms to transfer ubiquitin from E2 onto the target [10,11]. In addition to the catalytic domain(s), E3 ligases contain other domains [3,4,12,13,14] or form multi-subunit complexes [15, 16, 17, 18]. These ancillary components are generally involved in regulation or target recognition. It is becoming increasingly apparent that the ancillary domains and subunits are flexibly linked with conformational rearrangement underlying regulation.

Allosteric regulation provides a molecular means to transfer information in biological systems involving a protein and an effector. Allosteric effectors of the ubiquitination machinery can interact with either the catalytic domain or the ancillary domains to regulate enzymatic activity. Effectors are commonly separate molecules [19] but can also be regions of the substrate such as a growing ubiquitin chain [20, 21, 22]. We refer to these modes of regulation as acting in trans, that is, between separate molecules (Figure 2a). However, the multi-domain or multi-subunit architecture of many of these enzymes means effectors can also be regions of the enzyme itself [12,13,23]. We refer to this mode of regulation as acting in cis, that is, within the same molecule or complex (Figure 2b). Negative cis allostery appears to be a common method for auto-inhibition of E3 enzymes. An additional complication of allostery in ubiquitination is that the protein substrates themselves may exhibit allosteric changes driven by distinct effectors or the ubiquitination machinery itself. Finally, regulation can occur without any conformational changes via augmenting or steric hindrance of the substrate-binding interface, for example, via an adapter that interacts with both substrate and enzyme to promote substrate binding (Figure 2c). Although this may not be strictly classed as allostery, the binding sites for this type of effector are often distal to the active site due to the tendency for extended substrate-binding interfaces in the ubiquitination machinery. These modes may occur together, or in the context of oligomerization to regulate activity, as is the case for some RING and HECT E3s. Such a regulation of oligomeric states via an effector was termed polystery in the 1970s [24]. These features make the study of allostery in ubiquitination a complicated but exciting area.

Biological effectors of the ubiquitination machinery range from short polypeptide motifs to large protein surfaces. Ubiquitin itself has drawn special attention recently for its ability to regulate ubiquitination, which has been suggested as a mechanism to aid processive ubiquitination [20, 21, 22]. The role of ubiquitin in many protein–protein interactions has been exploited to generate ubiquitin variants (UbVs) that target-specific sites with high affinity using phage-display [22,25,26,27]. Here we examine recent examples of allosteric regulation of E2, RING-E3, HECT-E3, and RBR-E3 enzymes.

Section snippets

Ubiquitin-conjugating E2 enzymes and RING/U-box E3 ligases

Ubiquitin-conjugating E2s (∼35 members in humans) share a core ubiquitin conjugation (UBC) domain that bears a catalytic cysteine. They have a primary allosteric site where RING/U-box domains can bind to catalyse ubiquitin ligation onto the target lysine. Proteins comprising RING/U-box domains make up the largest family of ubiquitin E3 ligases (∼600 members in humans) and can be subgrouped into monomeric, homo-dimeric, hetero-dimeric, and cullin RING ligases (CRLs). In general, ancillary

HECT-type E3 ligases

For HECTs (∼28 members) the catalytic domain architecture consists of an N-lobe and C-lobe, which recruit E2∼Ub and contain a catalytic cysteine, respectively. Rearrangement of these lobes is necessary to transfer ubiquitin from the E2∼Ub onto the C-lobe catalytic cysteine via transthiolation and then onto the target. An allosteric-binding site for ubiquitin on the N-lobe has been shown to regulate activity probably through promoting chain elongation [20,21]. UbVs targeting HECTs revealed that

RBR-type E3 ligases

The RBR (RING Between RING) family of E3 ligases (14 members) comprise a tripartite arrangement of RING1, in-between RING (IBR) and RING2 domains. RING1 adopts a zinc-coordinating cross-brace typically observed in RING domains, while, the IBR and RING2 share a linear bilobal zinc-binding fold. The RBRs follow an obligate two-step catalytic cycle where the RING1, along with the IBR, first facilitate ubiquitin transthiolation between the E2∼Ub thioester and a catalytic cysteine in RING2 [49]. The

Conclusions

The ubiquitination machinery is made of complex enzymes often with multiple regulatory sites, effected in various ways (Table 1). Much progress has been made in identifying regulatory sites, in particular UbVs are a promising and general approach [25,26,27]. However, untangling the mechanisms of regulation is a non-trivial task as multiple mechanisms are seemingly at work, even for individual regulatory sites [12,23,33••,36••,45,47••]. The multiple steps of the ubiquitination pathway

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the EMBO Young Investigator Programme to H.W.; the European Research Council (ERC-2015-CoG-681582 ICLUb) consolidator grant to H.W. We thank members of the Walden laboratory and Dr Ciara Kyne for useful discussions.

References (59)

  • M. Hochstrasser

    Origin and function of ubiquitin-like proteins

    Nature

    (2009)
  • R. Yau et al.

    The increasing complexity of the ubiquitin code

    Nat Cell Biol

    (2016)
  • D. Komander et al.

    Breaking the chains: structure and function of the deubiquitinases

    Nat Rev Mol Cell Biol

    (2009)
  • P. Hanpude et al.

    Deubiquitinating enzymes in cellular signaling and disease regulation

    IUBMB Life

    (2015)
  • F.C. Streich et al.

    Capturing a substrate in an activated RING E3/E2-SUMO complex

    Nature

    (2016)
  • V.K. Chaugule et al.

    Specificity and disease in the ubiquitin system

    Biochem Soc Trans

    (2016)
  • J. Zhou et al.

    IUUCD 2.0: an update with rich annotations for ubiquitin and ubiquitin-like conjugations

    Nucleic Acids Res

    (2018)
  • N. Zheng et al.

    Ubiquitin ligases: structure, function, and regulation

    Annu Rev Biochem

    (2017)
  • Z. Chen et al.

    A tunable brake for HECT ubiquitin ligases

    Mol Cell

    (2017)
  • K. Zhu et al.

    Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch

    EMBO Rep

    (2017)
  • Y.J. Li et al.

    Allosteric inhibition of ubiquitin-like modifications by a class of inhibitor of SUMO-activating enzyme

    Cell Chem Biol

    (2019)
  • N.G. Brown et al.

    Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C

    Cell

    (2016)
  • M. Gorelik et al.

    A structure-based strategy for engineering selective ubiquitin variant inhibitors of Skp1-Cul1-F-box ubiquitin ligases

    Structure

    (2018)
  • M.E.R. Maitland et al.

    The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation

    Sci Rep

    (2019)
  • R. Das et al.

    Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

    EMBO J

    (2013)
  • E. Maspero et al.

    Structure of the HECT: ubiquitin complex and its role in ubiquitin chain elongati on

    EMBO Rep

    (2011)
  • H.C. Kim et al.

    Structure and function of a HECT domain ubiquitin-binding site

    EMBO Rep

    (2011)
  • E.R. Watson et al.

    Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

    Proc Natl Acad Sci U S A

    (2019)
  • Z. Wang et al.

    A multi-lock inhibitory mechanism for fine-tuning enzyme activities of the HECT family E3 ligases

    Nat Commun

    (2019)
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