Mitochondrial outer membrane integrity regulates a ubiquitin-dependent and NF-κB-mediated inflammatory response.

Mitochondrial outer membrane permeabilisation (MOMP) is often essential for apoptosis, by enabling cytochrome c release that leads to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory with emerging cellular roles, including its ability to elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell regulates this remains poorly defined. We find that upon MOMP, many proteins localised either to inner or outer mitochondrial membranes are ubiquitylated in a promiscuous manner. This extensive ubiquitylation serves to recruit the essential adaptor molecule NEMO, leading to the activation of pro-inflammatory NF-κB signalling. We show that disruption of mitochondrial outer membrane integrity through different means leads to the engagement of a similar pro-inflammatory signalling platform. Therefore, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-κB signalling.

PRL2 Phosphatase Promotes Oncogenic KIT Signaling in Leukemia Cells through Modulating CBL Phosphorylation.

Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function.

ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.

E3 ligase-inactivation rewires CBL interactome to elicit oncogenesis by hijacking RTK-CBL-CIN85 axis.

Casitas B-lineage lymphoma (CBL) is a ubiquitin ligase (E3) that becomes activated upon Tyr371-phosphorylation and targets receptor protein tyrosine kinases for ubiquitin-mediated degradation. Deregulation of CBL and its E3 activity is observed in myeloproliferative neoplasms and other cancers, including breast, colon, and prostate cancer. Here, we explore the oncogenic mechanism of E3-inactive CBL mutants identified in myeloproliferative neoplasms. We show that these mutants bind strongly to CIN85 under normal growth conditions and alter the CBL interactome. Lack of E3 activity deregulates CIN85 endosomal trafficking, leading to an altered transcriptome that amplifies signaling events to promote oncogenesis. Disruption of CBL mutant interactions with EGFR or CIN85 reduces oncogenic transformation. Given the importance of the CBL-CIN85 interaction in breast cancers, we examined the expression levels of CIN85, CBL, and the status of Tyr371-phosphorylated CBL (pCBL) in human breast cancer tissue microarrays. Interestingly, pCBL shows an inverse correlation with both CIN85 and CBL, suggesting that high expression of inactivated CBL could coordinate with CIN85 for breast cancer progression. Inhibition of the CBL-CIN85 interaction with a proline-rich peptide of CBL that binds CIN85 reduced the proliferation of MDA-MB-231 cells. Together, these results provide a rationale for exploring the potential of targeting the EGFR-CBL-CIN85 axis in CBL-inactivated mutant cancers.

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant.

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2's ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)-Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.

Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice.

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.

DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination.

Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-ribose-binding pocket and recruits poly-ADP-ribose (PAR)-modified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-ribose-binding domain that facilitates PAR-dependent ubiquitination.

Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain.

Phosphorylation of MDM2 by ATM upon DNA damage is an important mechanism for deregulating MDM2, thereby leading to p53 activation. ATM phosphorylates multiple residues near the RING domain of MDM2, but the underlying molecular basis for deregulation remains elusive. Here we show that Ser429 phosphorylation selectively enhances the ubiquitin ligase activity of MDM2 homodimer but not MDM2-MDMX heterodimer. A crystal structure of phospho-Ser429 (pS429)-MDM2 bound to E2-ubiquitin reveals a unique 310-helical feature present in MDM2 homodimer that allows pS429 to stabilize the closed E2-ubiquitin conformation and thereby enhancing ubiquitin transfer. In cells Ser429 phosphorylation increases MDM2 autoubiquitination and degradation upon DNA damage, whereas S429A substitution protects MDM2 from auto-degradation. Our results demonstrate that Ser429 phosphorylation serves as a switch to boost the activity of MDM2 homodimer and promote its self-destruction to enable rapid p53 stabilization and resolve a long-standing controversy surrounding MDM2 auto-degradation in response to DNA damage.

Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B∼ubiquitin complex.

Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes. Cellular inhibitor of apoptosis protein-1 (cIAP1) plays a central role in apoptosis and tumor necrosis factor signaling. It harbors a C-terminal RING domain that homodimerizes to recruit E2∼Ub (where ∼ denotes a thioester bond) complex to catalyze Ub transfer. Noncovalent Ub binding to the backside of the E2 Ub-conjugating enzyme UbcH5 has previously been shown to enhance RING domain activity, but the molecular basis for this enhancement is unclear. To investigate how dimeric cIAP1 RING activates E2∼Ub for Ub transfer and what role noncovalently bound Ub has in Ub transfer, here we determined the crystal structure of the cIAP1 RING dimer bound to both UbcH5B covalently linked to Ub (UbcH5B-Ub) and a noncovalent Ub to 1.7 Å resolution. The structure along with biochemical analyses revealed that the cIAP1 RING domain interacts with UbcH5B-Ub and thereby promotes the formation of a closed UbcH5B-Ub conformation that primes the thioester bond for Ub transfer. We observed that the noncovalent Ub binds to the backside of UbcH5B and abuts UbcH5B's α1ß1-loop, which, in turn, stabilizes the closed UbcH5B-Ub conformation. Our results disclose the mechanism by which cIAP1 RING dimer activates UbcH5B∼Ub and indicate that noncovalent Ub binding further stabilizes the cIAP1-UbcH5B∼Ub complex in the active conformation to stimulate Ub transfer.

A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity.

MDM2-MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2-MDMX-E2(UbcH5B)-ubiquitin complex, we designed MDM2 mutants that prevent E2-ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53's transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors.

Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation.

BACKGROUND: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR) between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl's conformation. Here, we investigate how Tyr371 mutations affect Cbl's conformation in solution and how this relates to Cbl's ability to potentiate transformation in cells. RESULTS: To explore how Tyr371 mutations affect Cbl's properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2-Ub), small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2-Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction. CONCLUSIONS: c-Cbl's LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl.

Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3.

RING E3 ligases catalyze the transfer of ubiquitin (Ub) from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to substrate. For RING E3 dimers, the RING domain of one subunit and tail of the second cooperate to prime Ub, but how this is accomplished by monomeric RING E3s in the absence of a tail-like component is currently unknown. Here, we present a crystal structure of a monomeric RING E3, Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2, revealing a similar mechanism in activating E2~Ub. Both pTyr363 and the pTyr363-induced element interact directly with Ub's Ile36 surface, improving the catalytic efficiency of Ub transfer by ~200-fold. Hence, interactions outside the canonical RING domain are crucial for optimizing Ub transfer in both monomeric and dimeric RING E3s. We propose that an additional non-RING Ub-priming element may be a common RING E3 feature.

BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer.

Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B∼Ub). The structure reveals extensive noncovalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub. Mutations that disrupt these noncovalent interactions or RING dimerization reduce UbcH5B∼Ub binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5B∼Ub induces peak-shift perturbations in the donor Ub consistent with the crystallographically-observed Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2∼Ub and optimize the donor Ub configuration for transfer.

Structural basis for autoinhibition and phosphorylation-dependent activation of c-Cbl.

Cbls are RING ubiquitin ligases that attenuate receptor tyrosine kinase (RTK) signal transduction. Cbl ubiquitination activity is stimulated by phosphorylation of a linker helix region (LHR) tyrosine residue. To elucidate the mechanism of activation, we determined the structures of human CBL, a CBL-substrate peptide complex and a phosphorylated-Tyr371-CBL-E2-substrate peptide complex, and we compared them with the known structure of a CBL-E2-substrate peptide complex. Structural and biochemical analyses show that CBL adopts an autoinhibited RING conformation, where the RING's E2-binding surface associates with CBL to reduce E2 affinity. Tyr371 phosphorylation activates CBL by inducing LHR conformational changes that eliminate autoinhibition, flip the RING domain and E2 into proximity of the substrate-binding site and transform the RING domain into an enhanced E2-binding module. This activation is required for RTK ubiquitination. Our results present a mechanism for regulation of c-Cbl's activity by autoinhibition and phosphorylation-induced activation.

Crystal structure of UBA2(ufd)-Ubc9: insights into E1-E2 interactions in Sumo pathways.

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2(ufd)), alone and in complex with Ubc9. The overall structures of both yeast Uba2(ufd) and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2(ufd)-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2(ufd)-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.