A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly.

Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental syndrome associated with a spectrum of developmental and intellectual delays and disabilities, harbor diverse mutations in WDR26, encoding a subunit of the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers of WDR26 bridge two core-CTLH E3 complexes to generate giant, hollow oval-shaped supramolecular CTLH E3 assemblies. Additionally, WDR26 mediates CTLH E3 complex binding to subunit YPEL5 and functions as substrate receptor for the transcriptional repressor HBP1. Here, we mapped SKDEAS-associated mutations on a WDR26 structural model and tested their functionality in complementation studies using genetically engineered human cells lacking CTLH E3 supramolecular assemblies. Despite the diversity of mutations, 15 of 16 tested mutants impaired at least one CTLH E3 complex function contributing to complex assembly and interactions, thus providing first mechanistic insights into SKDEAS pathology.

Structural insights into the functional mechanism of the ubiquitin ligase E6AP.

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.

Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase.

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.

PROTACable Is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning To Automate the De Novo Design of PROTACs.

Proteolysis-targeting chimeras (PROTACs) that engage two biological targets at once are a promising technology in degrading clinically relevant protein targets. Since factors that influence the biological activities of PROTACs are more complex than those of a small molecule drug, we explored a combination of computational chemistry and deep learning strategies to forecast PROTAC activity and enable automated design. A new method named PROTACable was developed for the de novo design of PROTACs, which includes a robust 3-D modeling workflow to model PROTAC ternary complexes using a library of E3 ligase and linker and an SE(3)-equivariant graph transformer network to predict the activity of newly designed PROTACs. PROTACable is available at https://github.com/giaguaro/PROTACable/.

Continuous evolution of compact protein degradation tags regulated by selective molecular glues.

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.

UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Molecular structure and function of mysterin/RNF213.

Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author performed the first molecular cloning of the mysterin gene as the final step in genetic exploration of cerebrovascular moyamoya disease (MMD) and initiated the next round of exploration to understand its molecular and cellular functions. Although much remains unknown, accumulating findings suggest that mysterin functions in cells by targeting massive intracellular structures, such as lipid droplets (LDs) and various invasive pathogens. In the latter case, mysterin appears to directly surround and ubiquitylate the surface of pathogens and stimulate cell-autonomous antimicrobial reactions, such as xenophagy and inflammatory response. To date, multiple mutations causing MMD have been identified within and near the RING finger domain of mysterin; however, their functional relevance remains largely unknown. Besides the RING finger, mysterin harbors a dynein-like ATPase core and an RZ finger, another ubiquitin ligase domain unique to mysterin, while functional exploration of these domains has also just commenced. In this review, the author attempts to summarize the core findings regarding the molecular structure and function of the mysterin protein, with an emphasis on the perspective of MMD research.

Simulation and Computational Study of RING Domain Mutants of BRCA1 and Ube2k in AD/PD Pathophysiology.

Lysine-based post-translational modification (PTM) such as acylation, acetylation, deamination, methylation, SUMOylation, and ubiquitination has proven to be a major regulator of gene expression, chromatin structure, protein stability, protein-protein interaction, protein degradation, and cellular localization. However, besides all the PTMs, ubiquitination stands as the second most common PTM after phosphorylation that is involved in the etiology of neurodegenerative diseases (NDDs) namely, Alzheimer's disease (AD) and Parkinson's disease (PD). NDDs are characterized by the accumulation of misfolded protein aggregates in the brain that lead to disease-related gene mutation and irregular protein homeostasis. The ubiquitin-proteasome system (UPS) is in charge of degrading these misfolded proteins, which involve an interplay of E1, E2, E3, and deubiquitinase enzymes. Impaired UPS has been commonly observed in NDDs and E3 ligases are the key members of the UPS, thus, dysfunction of the same can accelerate the neurodegeneration process. Therefore, the aim of this study is firstly, to find E3 ligases that are common in both AD and PD through data mining. Secondly, to study the impact of mutation on its structure and function. The study deciphered 74 E3 ligases that were common in both AD and PD. Later, 10 hub genes were calculated of which protein-protein interaction, pathway enrichment, lysine site prediction, domain, and motif analysis were performed. The results predicted BRCA1, PML, and TRIM33 as the top three putative lysine-modified E3 ligases involved in AD and PD pathogenesis. However, based on structural characterization, BRCA1 was taken further to study RING domain mutation that inferred K32Y, K32L, K32C, K45V, K45Y, and K45G as potential mutants that alter the structural and functional ability of BRCA1 to interact with Ube2k, E2-conjugating enzyme. The most probable mutant observed after molecular dynamics simulation of 50 ns is K32L. Therefore, our study concludes BRCA1, a potential E3 ligase common in AD and PD, and RING domain mutation at sites K32 and K45 possibly disturbs its interaction with its E2, Ube2k.

KH-like Domains in PARP9/DTX3L and PARP14 Coordinate Protein-Protein Interactions to Promote Cancer Cell Survival.

Certain members of the ADP-ribosyltransferase superfamily (ARTD or PARP enzymes) catalyse ADP-ribosylation in response to cellular stress, DNA damage and viral infection and are upregulated in various tumours. PARP9, its binding partner DTX3L and PARP14 protein levels are significantly correlated in head and neck squamous cell carcinoma (HNSCC) and other tumour types though a mechanism where PARP9/DTX3L regulates PARP14 post-transcriptionally. Depleting PARP9, DTX3L or PARP14 expression in HNSCC or HeLa cell lines decreases cell survival through a reduction of proliferation and an increase in apoptosis. A partial rescue of survival was achieved by expressing a PARP14 truncation containing a predicted eukaryotic type I KH domain. KH-like domains were also found in PARP9 and in DTX3L and contributed to protein-protein interactions between PARP9-DTX3L and PARP14-DTX3L. Homodimerization of DTX3L was also coordinated by a KH-like domain and was disrupted by site-specific mutation. Although, cell survival promoted by PARP14 did not require ADP-ribosyltransferase activity, interaction of DTX3L in vitro suppressed PARP14 auto-ADP-ribosylation and promoted trans-ADP-ribosylation of PARP9 and DTX3L. In summary, we characterised PARP9-DTX3L-PARP14 interactions important to pro-survival signalling in HNSCC cells, albeit in PARP14 catalytically independent fashion.

Allosteric Hsp70 Modulator YM-1 Induces Degradation of BRD4.

YM-1, an allosteric modulator of heat-shock 70 kDa protein (Hsp70), inhibits cancer cell growth, but the mechanism is not yet fully understood. Here, we show that YM-1 induces the degradation of bromodomain containing 4 (BRD4), which mediates oncogene expression. Overall, our results indicate that YM-1 promotes the binding of HSP70 to BRD4, and this in turn promotes the ubiquitination of BRD4 by C-terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase working in concert with Hsp70, leading to proteasomal degradation of BRD4. This YM-1-induced decrease of BRD4 would contribute at least in part to the inhibition of cancer cell growth.

The SDS22:PP1:I3 complex: SDS22 binding to PP1 loosens the active site metal to prime metal exchange.

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is ∼10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1's M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

The co-crystal structure of Cbl-b and a small-molecule inhibitor reveals the mechanism of Cbl-b inhibition.

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound's interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

DegronMD: Leveraging Evolutionary and Structural Features for Deciphering Protein-Targeted Degradation, Mutations, and Drug Response to Degrons.

Protein-targeted degradation is an emerging and promising therapeutic approach. The specificity of degradation and the maintenance of cellular homeostasis are determined by the interactions between E3 ubiquitin ligase and degradation signals, known as degrons. The human genome encodes over 600 E3 ligases; however, only a small number of targeted degron instances have been identified so far. In this study, we introduced DegronMD, an open knowledgebase designed for the investigation of degrons, their associated dysfunctional events, and drug responses. We revealed that degrons are evolutionarily conserved and tend to occur near the sites of protein translational modifications, particularly in the regions of disordered structure and higher solvent accessibility. Through pattern recognition and machine learning techniques, we constructed the degrome landscape across the human proteome, yielding over 18,000 new degrons for targeted protein degradation. Furthermore, dysfunction of degrons disrupts the degradation process and leads to the abnormal accumulation of proteins; this process is associated with various types of human cancers. Based on the estimated phenotypic changes induced by somatic mutations, we systematically quantified and assessed the impact of mutations on degron function in pan-cancers; these results helped to build a global mutational map on human degrome, including 89,318 actionable mutations that may induce the dysfunction of degrons and disrupt protein degradation pathways. Multiomics integrative analysis unveiled over 400 drug resistance events associated with the mutations in functional degrons. DegronMD, accessible at https://bioinfo.uth.edu/degronmd, is a useful resource to explore the biological mechanisms, infer protein degradation, and assist with drug discovery and design on degrons.

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes.

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural and single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

Resonance assignments of the PUB domain of the RNF31 protein.

E3 ubiquitin protein ligase RNF31 is present in human proteins and is involved in linear ubiquitin chain assembly complex (LUBAC) activity and cell growth. RNF31 is involved in ubiquitination, which is the post-translational modification of proteins. Ubiquitin molecules connect with amino acid residues of target proteins under the action of ubiquitin-activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, so as to achieve certain physiological functions. The abnormal expression of ubiquitination promotes the formation of cancer. In studies of breast cancer, RNF31 mRNA levels were found to be higher in cancer cells than in other tissues. The PUB domain of RNF31 is the binding site of the ubiquitin thioesterase otulin. Here, we report the backbone and side-chain resonance assignments of the PUB domain of RNF31 and study the backbone relaxation of the domain. These studies will contribute to further understanding of the structural and functional relationship of RNF31 protein, which may also be a target for drug research.

New insights into cancer: MDM2 binds to the citrullinating enzyme PADI4.

PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens.