Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5.

Ubiquitin (Ub) chain formation by homologous to E6AP C-terminus (HECT)-family E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We used chemistry and cryo-electron microscopy (cryo-EM) to visualize stable mimics of the intermediates along K48-linked Ub chain formation by the human E3, UBR5. The structural data reveal a ≈ 620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly tethered Ub-associated (UBA) domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor Ub, with its K48 lured into the active site by numerous interactions between the acceptor Ub, manifold UBR5 elements and the donor Ub. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing Ub transfer from E2 to E3 and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2 and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked Ub chain forging machine.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

Chemical Synthesis of Non-hydrolyzable Ubiquitin(-Like) Hybrid Chains.

Hybrid chains are a combination of ubiquitin (Ub) and Ub-like (UbL) proteins, expanding on the finely tuned Ub code. To decipher this intricate code, understanding of its assembly, architecture, as well as specific interactors of these Ub/UbL hybrid chains are important, warranting the development of suitable reagents. Here, we describe the chemical methodology to access linkage specific non-hydrolyzable Ub-NEDD8-based chains endowed with an affinity handle in all possible combinations of K48 hybrid chain dimers between Ub and NEDD8.

Hybrid Chains: A Collaboration of Ubiquitin and Ubiquitin-Like Modifiers Introducing Cross-Functionality to the Ubiquitin Code.

The Ubiquitin CODE constitutes a unique post-translational modification language relying on the covalent attachment of Ubiquitin (Ub) to substrates, with Ub serving as the minimum entity to generate a message that is translated into different cellular pathways. The creation of this message is brought about by the dedicated action of writers, erasers, and readers of the Ubiquitin CODE. This CODE is greatly expanded through the generation of polyUb chains of different architectures on substrates thus regulating their fate. Through additional post-translational modification by Ub-like proteins (UbL), hybrid Ub/UbL chains, which either alter the originally encrypted message or encode a completely new one, are formed. Hybrid Ub/UbL chains are generated under both stress or physiological conditions and seem to confer improved specificity and affinity toward their cognate receptors. In such a manner, their formation must play a specific, yet still undefined role in cellular signaling and thus understanding the UbCODE message is crucial. Here, we discuss the evidence for the existence of hybrid Ub/UbL chains in addition to the current understanding of its biology. The modification of Ub by another UbL complicates the deciphering of the spatial and temporal order of events warranting the development of a hybrid chain toolbox. We discuss this unmet need and expand upon the creation of tailored tools adapted from our previously established toolkit for the Ubiquitin Proteasome System to specifically target these hybrid Ub/UbL chains.