Building Ubiquitination Machineries: E3 Ligase Multi-Subunit Assembly and Substrate Targeting by PROTACs and Molecular Glues
Introduction
The ubiquitin-proteasome system (UPS) is a central regulator of protein homeostasis and is increasingly recognized as a promising therapeutic target due to its involvement in diseases such as cancer and neurodegeneration. The post-translational addition of ubiquitin to substrate proteins is orchestrated by a cascade involving three enzymes: the E1-activating enzyme, which activates ubiquitin (Ub) in an ATP-dependent manner; the E2-conjugating enzyme, which receives the activated Ub via trans-esterification; and the E3 ubiquitin ligase, which catalyzes the transfer of Ub from the E2 to a lysine residue on the substrate, typically forming an isopeptide bond, though esterification on threonine residues has also been reported.
E3 ubiquitin ligases are classified into three main classes: homologous to E6-AP C-terminus (HECT), really interesting new gene (RING), and RING-between-RING (RBR) ligases. HECT E3s form a covalent thioester intermediate with Ub before transferring it to the substrate. RING E3s facilitate the direct transfer of Ub from E2~Ub to the substrate by bringing them into proximity. RBR ligases combine features of both HECT and RING families, first recruiting E2~Ub and then transferring Ub to a HECT-type catalytic cysteine before the final transfer to the substrate. The anaphase-promoting complex (APC/C) is a large assembly of 11–13 proteins, including a cullin (Apc2) and RING (Apc11) subunit, and plays a key role in cell cycle regulation.
E3 ligases are central to substrate specificity. Their ubiquitination activity on native substrates is tightly regulated by protein-protein interactions (PPI) that dictate their structural assembly. Additionally, small-molecule degraders such as molecular glues and proteolysis-targeting chimeras (PROTACs) can recruit non-native proteins to E3 ligases, hijacking their catalytic activity to target neo-substrates for proteasomal degradation. This review focuses on recent advances in understanding the structural basis of building and hijacking ubiquitination machineries, with an emphasis on Cullin RING E3 ligase assembly, substrate recognition, and substrate recruitment by degraders, which hold significant therapeutic potential.
Structural Assembly and Activity of Modular Multi-Subunit E3 Ligases
Cullin RING E3 ubiquitin ligases (CRLs) represent the largest family of E3 ligases. They are modular, composed of interchangeable substrate receptors, adaptor subunits, and a RING-box domain subunit, all assembled around a central cullin scaffold. CRLs are classified based on the type of cullin subunit (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7). Structures of fully assembled CRL complexes have revealed a range of conformations and orientations among different cullin subunits.
The crystal structure of the CRL2VHL complex, which includes Cul2, RBX1, Elongin B, Elongin C, and von Hippel-Lindau protein (VHL), highlights inherent interdomain flexibility in cullin scaffold proteins. This flexibility allows the cullin C-terminal globular domain and N-terminal helical bundles to come closer together compared to other CRL structures. The RBX1 RING domain is captured in an intermediate state between inactive and NEDD8-activated conformations. NEDD8 is a ubiquitin-like protein whose post-translational modification of cullins is essential for CRL activation.
A recent cryo-electron microscopy structure captured a neddylated CRL1B-TRCP complex in an intermediate state en route to substrate ubiquitination. This structure comprises neddylated CRL1B-TRCP, ubiquitin-loaded E2 enzyme UBE2D, and a phosphorylated peptide from IκBα. Three modules are evident: the activation module (NEDD8 covalently linked to cullin), the catalytic module (UBE2D~Ub and RBX1), and the substrate-scaffolding module (substrate receptor β-TrCP, SKP1, and cullin). The mobile WHB domain of cullin enables multiple contacts that bring the catalytic center of UBE2D into proximity with the β-TrCP-bound substrate for ubiquitination.
Neddylation is reversible, with deneddylation catalyzed by the COP9 signalosome (CSN), an eight-subunit complex. Cryo-EM structures of CSN bound to neddylated CRLs provide detailed insights into the conserved activation mechanism of deneddylation, including conformational clamping of CRL by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer, and activation of CSN5 metalloprotease activity. Inositol hexakisphosphate (IP6) has been identified as a CSN cofactor that enhances interaction with RBX1 and mediates CSN sequestration of CRL4, preventing its activation.
The eukaryotic N-end rule pathway, which governs protein degradation based on N-terminal residues, has been expanded to include the Pro/N-degron branch, recognized by GID4, a subunit of the GID assembly ubiquitin ligase. GID is a multisubunit E3 ligase from yeast that recognizes the N-terminal proline of gluconeogenic enzymes and catalyzes their ubiquitination. The GID assembly transitions from an anticipatory state under carbon stress to an active state upon glucose recovery, recognizing and ubiquitinating specific substrates.
Substrate Recognition by E3 Ligase Substrate Receptors
CRLs utilize substrate receptor modules to provide specificity for substrate recognition. Recent advances have illuminated the structural basis of this diversity. The CRL1FBXL5–iron regulatory protein 2 (IRP2) complex, for example, reveals how the [2Fe2S] cluster in FBXL5 mediates oxygen sensing and substrate binding. The CRL2 subunit KLHDC2 recognizes substrates via a novel C-end degron mechanism, with the Kelch domain forming a deep pocket that binds the substrate’s terminal carboxyl group with high affinity.
CRL3 substrate receptors combine substrate binding and adaptor domains in a single polypeptide. Kelch-like proteins such as KLHL12 and KLHL20 recognize specific degron motifs in their substrates, with structural studies revealing how substrate peptides adopt unique conformations to fit into the Kelch domain pockets. The MATH domain of SPOP, another CRL3 substrate receptor, binds a variety of substrates, relaxing the consensus binding motif.
CRL5 substrate-bound structures, including those of suppressor of cytokine signaling proteins (SOCS1, SOCS2) and ankyrin repeat and SOCS box protein 9 (ASB9), have recently been solved. SOCS1 and SOCS2 share a domain architecture with an N-terminal extended SH2 subdomain, a central SH2 domain for phosphotyrosine recognition, and a SOCS box for interaction with the ElonginB-ElonginC adaptor complex. SOCS1 also contains a kinase inhibitory region (KIR) that blocks the substrate binding groove of Janus kinases, while SOCS2 uses its SH2 domain to recognize phosphodegrons from erythropoietin and growth hormone receptors.
PROTACs: Bifunctional Small Molecules Bridging Target Proteins to E3 Ligases
PROteolysis TArgeting Chimeras (PROTACs) are bifunctional degrader molecules composed of an E3 ligase ligand and a target protein ligand, joined by a chemical linker. PROTACs can independently bind to the E3 ligase or the target protein (forming 1:1 complexes) before inducing proximity between the two proteins in a ternary complex (1:1:1). Unlike molecular glues, which lack a linker and can only bind one of the two proteins, PROTACs were initially thought to function independently of PPIs between the ligase and the target. However, structural and biophysical studies have revealed that PROTACs can also ‘glue’ E3 ligase and target protein into stable and cooperative ternary complexes.
The first PROTAC ternary structure solved involved MZ1, a degrader of the BET protein Brd4, bound to VHL–ElonginC–ElonginB and the second bromodomain of Brd4. The crystal structure revealed the formation of a stable ternary complex, with the PROTAC acting as a molecular bridge. Structural studies of various PROTACs have highlighted the plasticity of the E3–target interface and suggest that even suboptimal ternary complexes can be productive for targeted protein degradation if composed of high-affinity ligands.
Molecular Glues and Ternary Complexes
Molecular glues are small molecules that stabilize the interaction between an E3 ligase and a substrate, promoting ubiquitination and degradation. Unlike PROTACs, molecular glues do not contain a linker and bind to only one partner, but induce or stabilize a PPI interface that recruits the other. Structural studies of E3 ligases bound to molecular glues and neo-substrates, such as thalidomide analogs with CRBN and aryl-sulfonamides with DCAF15, have provided valuable insights into the mechanisms of action and have informed structure-based design of new degraders.
Conclusions
Recent developments in the structural understanding of E3 ligase assembly, function, and substrate recognition have underscored the importance of these complexes in regulating protein homeostasis. With over 600 E3 ligases in mammals, their role in fine-tuning substrate specificity is critical. E3 ligases are emerging as attractive drug targets, and knowledge of substrate-bound structures can guide the development of small-molecule inhibitors and degraders. The advent of protein degraders that hijack E3 ligase activity to target disease-driving proteins is motivating further research into this protein family. Structural and biophysical characterization of E3 ligase ternary complexes is increasingly important for degrader drug design, with the potential to expand therapeutic options through cell-, tissue-, and disease-specific SJ6986 targeting.