Molecular glue CELMoD compounds are regulators of cereblon conformation.

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.

Allosteric differences dictate GroEL complementation of E. coli.

GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.

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.

Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

Gene expression and cell identity controlled by anaphase-promoting complex.

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2-5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.

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.

Posing the APC/C E3 Ubiquitin Ligase to Orchestrate Cell Division.

The anaphase promoting complex/cyclosome (APC/C) E3 ligase controls mitosis and nonmitotic pathways through interactions with proteins that coordinate ubiquitylation. Since the discovery that the catalytic subunits of APC/C are conformationally dynamic cullin and RING proteins, many unexpected and intricate regulatory mechanisms have emerged. Here, we review structural knowledge of this regulation, focusing on: (i) coactivators, E2 ubiquitin (Ub)-conjugating enzymes, and inhibitors engage or influence multiple sites on APC/C including the cullin-RING catalytic core; and (ii) the outcomes of these interactions rely on mobility of coactivators and cullin-RING domains, which permits distinct conformations specifying different functions. Thus, APC/C is not simply an interaction hub, but is instead a dynamic, multifunctional molecular machine whose structure is remodeled by binding partners to achieve temporal ubiquitylation regulating cell division.

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.

Measuring APC/C-Dependent Ubiquitylation In Vitro.

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination.

Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli3(1-90)) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3(SPOP) and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli3(1-90), the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli3(1-90) ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2∼Ub catalytic modules such as APC11-UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10∼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

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.

SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer.

Hypoxic stress and hypoxia-inducible factors (HIFs) play important roles in a wide range of tumors. We demonstrate that SPOP, which encodes an E3 ubiquitin ligase component, is a direct transcriptional target of HIFs in clear cell renal cell carcinoma (ccRCC). Furthermore, hypoxia results in cytoplasmic accumulation of SPOP, which is sufficient to induce tumorigenesis. This tumorigenic activity occurs through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis, including the tumor suppressor PTEN, ERK phosphatases, the proapoptotic molecule Daxx, and the Hedgehog pathway transcription factor Gli2. Knockdown of SPOP specifically kills ccRCC cells, indicating that it may be a promising therapeutic target. Collectively, our results indicate that SPOP serves as a regulatory hub to promote ccRCC tumorigenesis.

Electron microscopy structure of human APC/C(CDH1)-EMI1 reveals multimodal mechanism of E3 ligase shutdown.

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C(CDH1) during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C(CDH1) functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C(CDH1) to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.

Complex interactions of Tyrp1 in the eye.

PURPOSE: To use a systems genetics approach to construct and analyze co-expression networks that are causally linked to mutations in a key pigementation gene, tyrosinase-related protein 1 (Tyrp1), that is associated both with oculocutaneous albinism type 3 (OCA3) in humans and with glaucoma in mice. METHODS: Gene expression patterns were measured in whole eyes of a large family of BXD recombinant inbred (RI) mice derived from parental lines that encode for wildtype (C57BL/6J) and mutant (DBA/2J) Tyrp1. Protein levels of Tyrp1 were measured in whole eyes and isolated irides. Bioinformatics analyses were performed on the expression data along with our archived sequence data. Separate data sets were generated which were comprised of strains that harbor either wildtype or mutant Tyrp1 and each was mined individually to identify gene networks that covary significantly with each isoform of Tyrp1. Ontology trees and network graphs were generated to probe essential function and statistical significance of covariation. Genes with strong covariance in wildtype mice were assembled into genome-wide heatmaps for cohorts carrying either wildtype or mutant Tyrp1. RESULTS: Single nucleotide polymorphism (SNP) analysis verified the presence of the Tyrp1b mutation in the Tyrp1 gene. Message levels were greater in BXD strains with the mutant Tyrp1. Interval mapping of these BXD mice revealed a strong expression quantitative trait locus (eQTL) on Chr 4 at the location of the gene itself. Composite mapping revealed a suggestive eQTL on Chr 9 at the location of myosin-Va (Myo5a), mutations in which are known as dilute. Enriched biologic processes associated with wildtype Tyrp1 included pigmentation, melanin biosynthetic process, and mesenchymal cell development, while associations with the mutant gene included categories of neural crest cell development, protein metabolic processes and glycoprotein metabolic processes. Genome-wide heatmaps revealed strong candidate cis-eQTLs on Chr 4 at Tyrp1 and on Chr 9 at Myo5a in all mice. In the wildtype data set, Tyrp1 was an upstream regulator of six pigmentation and two mesenchyme genes. In addition, five genes, including Tyrp1, were at least partially regulated by Myo5a. Analyses of the strains harboring the mutant gene revealed significant loss of correlation to traditional genes and gain of correlation to genes with little or no functional relationship. CONCLUSIONS: These findings indicate that the Tyrp1(b) mutation modifies the pathways and gene networks in which Tyrp1 functions. Our results also indicate direct and indirect regulatory control of Tyrp1 and other pigmentation and mesenchymal genes by Myo5a. Lastly, we find that the mutations reduce the ability of Tyrp1 to regulate expression of other genes that participate in pigmentation metabolism.