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.

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.

A Comprehensive Analysis of the Structural Recognition between KCTD Proteins and Cullin 3.

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

Cullin-associated and neddylation-dissociated 1 regulate reprogramming of lipid metabolism through SKP1-Cullin-1-F-boxFBXO11 -mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma.

BACKGROUND: Enhanced de novo lipogenesis is essential for hepatocellular carcinoma (HCC). Abnormally high cullin-associated and neddylation-dissociated 1 (CAND1) expression is associated with poor clinical prognosis in HCC. The SKP1-Cullin-1-F-box (SCF) complex consists of the SKP1, Cullin-1 and F-box proteins (FBPs) and performs multiple functions including adipogenesis. SCF complex was modulated by CAND1, but Whether and how the CAND1 promotes HCC by regulating SCF complex and lipogenesis are unknown. METHODS: HCC samples were used to analyze the correlations between CAND1 expression and clinicopathological characteristics such as survival and prognosis. The in vitro functions of CAND1, FBXO11 and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) were measured by cell proliferation, colony formation and migration assays. The in vivo functions were tested in multiple mouse liver cancer models including patient-derived xenograft (PDX), cell line-derived xenograft and AKT/NRASV12-induced primary liver cancer models. Injections of adeno-associated virus targeting CAND1 (AAV-shCAND1) were performed to evaluate the therapeutic efficacy of targeting CAND1. RNA-Seq and lipidomic assays followed by serial biochemical experiments including mass spectrometry, immunoprecipitation and GST pull-down were performed to dissect the underlying mechanisms. RESULTS: CAND1 promoted the expression of lipid synthesis genes by disrupting SCF complex assembly and lipid accumulation. Furthermore, we identified hnRNPA2B1 as a novel F-box protein 11 (FBXO11)-binding partner. FBXO11 directly bound to hnRNPA2B1 and promoted hnRNPA2B1 ubiquitination and subsequent degradation. Our evaluations of the therapeutic efficacy of AAV-shCAND1 injections confirmed that targeting the CAND1-SCFFBXO11 -hnRNPA2B1A signalling axis was therapeutically effective. CAND1 downregulation significantly reduced the tumour burden in a primary mouse liver cancer model and a PDX model. CONCLUSIONS: Our results highlight that CAND1 is associated with poor prognosis in HCC and regulates lipid metabolic reprogramming by dissociating the SCF complex. Targeting the CAND1-SCFFBXO11 -hnRNPA2B1 axis may be a novel strategy for HCC treatment.

Structural basis for the ubiquitination of G protein ßγ subunits by KCTD5/Cullin3 E3 ligase.

G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gßγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gßγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gßγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gßγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gßγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gßγ subunits. Moreover, we show that ubiquitination of Gßγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.

Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.

Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.

Review: A silent concert in developing plants: Dynamic assembly of cullin-RING ubiquitin ligases.

Plants appear quiet: quietly, they break the ground, expand leaves, search for resources, alert each other to invaders, and heal their own wounds. In contrast to the stationary appearance, the inside world of a plant is full of movements: cells divide to increase the body mass and form new organs; signaling molecules migrate among cells and tissues to drive transcriptional cascades and developmental programs; macromolecules, such as RNAs and proteins, collaborate with different partners to maintain optimal organismal function under changing cellular and environmental conditions. All these activities require a dynamic yet appropriately controlled molecular network in plant cells. In this short review, we used the regulation of cullin-RING ubiquitin ligases (CRLs) as an example to discuss how dynamic biochemical processes contribute to plant development. CRLs comprise a large family of modular multi-unit enzymes that determine the activity and stability of diverse regulatory proteins playing crucial roles in plant growth and development. The mechanism governing the dynamic assembly of CRLs is essential for CRL activity and biological function, and it may provide insights and implications for the regulation of other dynamic multi-unit complexes involved in fundamental processes such as transcription, translation, and protein sorting in plants.

Structure of CRL7FBXW8 reveals coupling with CUL1-RBX1/ROC1 for multi-cullin-RING E3-catalyzed ubiquitin ligation.

Most cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7FBXW8 assembly. CUL7's exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1-FBXW8 to a neddylated CUL1-RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL-CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.

Cullin-associated and neddylation-dissociated 1 protein (CAND1) governs cardiac hypertrophy and heart failure partially through regulating calcineurin degradation.

Pathological cardiac hypertrophy is a process characterized by significant disturbance of protein turnover. Cullin-associated and Neddylation-dissociated 1 (CAND1) acts as a coordinator to modulate substrate protein degradation by promoting the formation of specific cullin-based ubiquitin ligase 3 complex in response to substrate accumulation, which thereby facilitate the maintaining of normal protein homeostasis. Accumulation of calcineurin is critical in the pathogenesis of cardiac hypertrophy and heart failure. However, whether CAND1 titrates the degradation of hypertrophy related protein eg. calcineurin and regulates cardiac hypertrophy remains unknown. Therefore, we aim to explore the role of CAND1 in cardiac hypertrophy and heart failure and the underlying molecular mechanism. Here, we found that the protein level of CAND1 was increased in cardiac tissues from heart failure (HF) patients and TAC mice, whereas the mRNA level did not change. CAND1-KO+ /- aggravated TAC-induced cardiac hypertrophic phenotypes; in contrast, CAND1-Tg attenuated the maladaptive cardiac remodeling. At the molecular level, CAND1 overexpression downregulated, whereas CAND1-KO+ /- or knockdown upregulated calcineurin expression at both in vivo and in vitro conditions. Mechanistically, CAND1 overexpression favored the assembly of Cul1/atrogin1/calcineurin complex and rendered the ubiquitination and degradation of calcineurin. Notably, CAND1 deficiency-induced hypertrophic phenotypes were partially rescued by knockdown of calcineurin, and application of exogenous CAND1 prevented TAC-induced cardiac hypertrophy. Taken together, our findings demonstrate that CAND1 exerts a protective effect against cardiac hypertrophy and heart failure partially by inducing the degradation of calcineurin.

Kelch-like protein 3 in human disease and therapy.

Kelch-like protein 3 (KLHL3) is a substrate adaptor of Cullin3-RING ubiquitin ligase (CRL3), and KLHL3-CUL3 complex plays a vital role in the ubiquitination of specific substrates. Mutations and abnormal post-translational modifications of KLHL3-CUL3 affect substrate ubiquitination and may related to the pathogenesis of Gordon syndrome (GS), Primary Hyperparathyroidism (PHPT), Diabetes Mellitus (DM), Congenital Heart Disease (CHD), Pre-eclampsia (PE) and even cancers. Therefore, it is essential to understand the function and molecular mechanisms of KLHL3-CUL3 for the treatment of related diseases. In this review, we summary the structure and function of KLHL3-CUL3, the effect of KLHL3-CUL3 mutations and aberrant modifications in GS, PHPT, DM, CHD and PE. Moreover, we noted a possible role of KLHL3-CUL3 in carcinogenesis and provided ideas for targeting KLHL3-CUL3 for related disease treatment.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination.

As the largest family of ubiquitin (Ub) E3 ligases, cullin-RING ligases (CRLs) play crucial roles in various cellular processes, and their activities are tightly regulated by orchestrated mechanisms. Neddylation, the conjugation of a Ub-like protein NEDD8 to a target protein such as the cullin, represents a key regulatory mechanism for CRLs. Biochemical and structural studies of a few CRLs have revealed that cullin neddylation alters the CRL conformation and activates CRL-dependent protein ubiquitination. Here, using CUL2-RING ligase (CRL2) as an example, we describe our protocols for the preparation of recombinant CUL2 with or without NEDD8 conjugation, which is further used to quantitatively determine the effect of neddylation on CRL2-dependent protein ubiquitination in vitro. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of CUL2•RBX1 from Escherichia coli Support Protocol: Further purification of CUL2•RBX1 with additional chromatography on an FPLC system Basic Protocol 2: Reconstitution of cullin neddylation for quantitative ubiquitination assay in vitro.

Plant systemic acquired resistance compound salicylic acid as a potent inhibitor against SCF (SKP1-CUL1-F-box protein) mediated complex in Fusarium oxysporum by homology modeling and molecular dynamics simulations.

Fusarium oxysporum causes significant economic losses in many crop plants by causing root rot, necrosis, and wilting symptoms. Homology and molecular dynamics studies are promising tools for the detection in F. oxysporum of the systemic resistance compound, salicylic acid, for control of the SKP1-CUL1-F-box protein complex. The structure of SKP1-CUL1-F-box subunit Skp1 from F. oxysporum is produced by Modeler 9v7 for the conduct of docking studies. The Skp1 structure is based on the yeast Cdc4/Skp1 (PDB ID: 3MKS A) crystal structure collected by the Protein data bank. Applying molecular dynamic model simulation methods to the final predicted structure and further evaluated by 3D and PROCHECK test programmers, the final model is verified to be accurate. Applying GOLD 3.0.1, SCF Complex Skp1 is used to prevent stress-tolerant operation. The SKP1-CUL1-F-box model is predicted to be stabilized and tested as a stable docking structure. The predicted model of the SCF structure has been stabilized and confirmed to be a reliable structure for docking studies. The results indicated that GLN8, LYS9, VAL10, TRP11, GLU48, ASN49 in SCF complex are important determinant residues in binding as they have strong hydrogen bonding with salicylic acid, which showed best docking results with SKP1-CUL1-F-box complex subunit Skp1 with docking score 25.25KJ/mol. Insilco studies have been used to determine the mode of action of salicylic acid for Fusarium control. Salicylic acid hinders the SKP1-CUL1-F-box complex, which is important in protein-like interactions through hydrogen bodings. Results from docking studies have shown that the best energy for SKP1-CUL1-F-box was salicylic acid.Communicated by Ramaswamy H. Sarma.

NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics.

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αß subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.

Co-operative binding of SKP1, Cullin1 and Cullin7 to FBXW8 results in Cullin1-SKP1-FBXW8-Cullin7 functional complex formation that monitors cellular function of ß-TrCP1.

F-box protein FBXW8 is known to interact with scaffolding protein Cullin1 and Cullin7 to form SCF (SKP1, Cullin and F-box protein) complex. However, detail understanding about the importance of both Cullins for SCF-FBXW8 complex formation as well as its ubiquitin ligase activity remains elusive. Here, we show that, through in vitro and in vivo studies, Cullin1 and Cullin7 increase each other's binding to FBXW8 synergistically. Interestingly, absence of either Cullin results in abrogation of binding of other Cullin to FBXW8. Binding of SKP1 to FBXW8 also increases in the presence of both the Cullins. Thus, SKP1, Cullin1 and Cullin7 are essential to form Cullin1-SKP1-FBXW8-Cullin7 functional ubiquitin ligase complex. Further, using computational, mutational and biochemical analysis, we found that Cullin1 binds to N-terminus of FBXW8 through SKP1 while Cullin7 associates with C-terminus of FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex in a cooperative manner. Results showed that Cullin1-SKP1-FBXW8-Cullin7 complex plays a key role in maintaining the basal level expression of ß-TrCP1. Moreover, Cullin1-SKP1-FBXW8-Cullin7 complex promotes cell migration by activating ß-catenin via directing proteasomal degradation of ß-TrCP1. Overall, our study reveals the intriguing molecular mechanism of assembly of SKP1, Cullin1, Cullin7 and FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex that control the function of ß-TrCP1.

SPT5 stabilization of promoter-proximal RNA polymerase II.

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses.

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

Characterization of an A3G-VifHIV-1-CRL5-CBFß Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFß) to determine the structure of the (A3G-Vif-CRL5-CBFß) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFß complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Cullin-RING Ubiquitin Ligase Regulatory Circuits: A Quarter Century Beyond the F-Box Hypothesis.

Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.

The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases.

Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.