Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5.

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of human UBR5, revealing an α-solenoid scaffold with numerous protein-protein interacting motifs, assembled into an antiparallel dimer that adopts further oligomeric states. Using cryo-EM processing tools, we observe the dynamic nature of the UBR5 catalytic domain, which we postulate is important for its enzymatic activity. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates and several distinct domains for protein-protein interactions may explain how UBR5 is linked to several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3 ligases.

Dosage sensitivity to Pumilio1 variants in the mouse brain reflects distinct molecular mechanisms.

Different mutations in the RNA-binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA-binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1-specific targets, but the severe mutation disrupts interactions with several RNA-binding proteins and the regulation of their targets. In patient-derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA-binding proteins in a physiological context will require studying their interactions as well as their targets.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Friend or foe? Reciprocal regulation between E3 ubiquitin ligases and deubiquitinases.

Protein ubiquitination is a post-translational modification that entails the covalent attachment of the small protein ubiquitin (Ub), which acts as a signal to direct protein stability, localization, or interactions. The Ub code is written by a family of enzymes called E3 Ub ligases (∼600 members in humans), which can catalyze the transfer of either a single ubiquitin or the formation of a diverse array of polyubiquitin chains. This code can be edited or erased by a different set of enzymes termed deubiquitinases (DUBs; ∼100 members in humans). While enzymes from these distinct families have seemingly opposing activities, certain E3-DUB pairings can also synergize to regulate vital cellular processes like gene expression, autophagy, innate immunity, and cell proliferation. In this review, we highlight recent studies describing Ub ligase-DUB interactions and focus on their relationships.

Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase.

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

Insight into Viral Hijacking of CRL4 Ubiquitin Ligase through Structural Analysis of the pUL145-DDB1 Complex.

Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and key regulator of cell cycle progression. Since APC/C promotes the degradation of mitotic cyclins, it controls cell cycle-dependent oscillations in cyclin-dependent kinase (CDK) activity. Both CDKs and APC/C control a large number of substrates and are regulated by analogous mechanisms, including cofactor-dependent activation. However, whereas substrate dephosphorylation is known to counteract CDK, it remains largely unknown whether deubiquitinating enzymes (DUBs) antagonize APC/C substrate ubiquitination during mitosis. Here, we demonstrate that Cezanne/OTUD7B is a cell cycle-regulated DUB that opposes the ubiquitination of APC/C targets. Cezanne is remarkably specific for K11-linked ubiquitin chains, which are formed by APC/C in mitosis. Accordingly, Cezanne binds established APC/C substrates and reverses their APC/C-mediated ubiquitination. Cezanne depletion accelerates APC/C substrate degradation and causes errors in mitotic progression and formation of micronuclei. These data highlight the importance of tempered APC/C substrate destruction in maintaining chromosome stability. Furthermore, Cezanne is recurrently amplified and overexpressed in numerous malignancies, suggesting a potential role in genome maintenance and cancer cell proliferation.

Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase that initiates anaphase and mitotic exit. APC/C is activated by Cdc20 and inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin as a small molecule ligand of Cdc20 that inhibits APC/CCdc20 and prolongs mitosis. Here we find that apcin paradoxically shortens mitosis when SAC activity is high. These opposing effects of apcin arise from targeting of a common binding site in Cdc20 required for both substrate ubiquitination and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context.

Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly.

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding.

Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition.

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

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.

Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Advanced ERCP techniques for the extraction of complex biliary stones: a single referral center's 12-year experience.

OBJECTIVES: Advanced ERCP techniques (AETs) for difficult biliary stones include peroral cholangioscopy (POC) with electrohydraulic/laser lithotripsy (EHL/LL), endoscopic papillary large balloon dilation (EPLBD) and mechanical lithotripsy (ML). We assess the efficacy of AETs. METHODS: A retrospective query for AETs. PRIMARY OUTCOME: Complete duct clearance. Secondary outcome: Complete duct clearance by technique. Statistical Analysis version 9.3 (SAS Inc., Cary, NC). RESULTS: From 1/00 to 10/12, 349 patients were identified of which 222 (80% had prior ERCPs) had AETs. 211 with sufficient follow-up underwent 295 ERCPs; 280 of which were AET's. Index AETs: POC with EHL/LL (n = 46/211, 22%), ML (n = 84/211, 40%), EPLBD with mean balloon size of 11.5 ± 1.7 mm (n = 39/211, 18%) and combination AETs (n = 42/211, 20%). Stone characteristics: 76% had ≥1 stone, 81% extrahepatic and 32% had strictures. Number of stones (mean 2.5 and range 1-20) did not differ among groups. EPLBD had higher percentage (95%) of extrahepatic stones (p = .0003). The 'Combination' and 'POC' groups had larger stones (mean 17.7 mm ±6.4 and 16.8 mm ±6.1, respectively; p < .001). Complete clearance: 209/211 (99%) at index AET 167/211 (79%) or after mean of 2.5 ± 0.7 AETs in 42/211 (20%). Partial clearance: 2/211 (1%). Clearance at index AETs was higher with EPLBD (90%, p = .014). Adverse Events: 7/280 (2.5%). CONCLUSIONS: AETs achieved clearance in 99%. EPLBD had higher clearance at index AET likely owing to higher extrahepatic stones. Larger stones, but not number, were associated with increased combination AETs and total ERCPs.