15420117
RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex
Significance The anaphase-promoting complex/cyclosome (APC) is a multisubunit RING E3 ubiquitin (Ub) ligase that regulates mitosis, meiosis, and numerous facets of neurobiology by targeting key regulatory proteins for Ub-mediated degradation. Despite great importance, it remains unclear how APC, or most of the other 600 RING E3s in humans, targets Ub to lysines in disordered substrates. Here, we report the structural and molecular basis for substrate ubiquitination by APC and its partner E2, UBCH10. UBCH10 is recruited to APC, activated for ubiquitination, and positioned for substrate targeting through multisite interactions with the APC cullin–RING core. We propose that many RING E3–E2 assemblies work similarly, with multisite interactions establishing specificity, harnessing ubiquitination machineries to accelerate searching for target lysines, and facilitating regulation. 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 APCCDH1 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 APCCDH1–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.
35042803
RING domain E3 ubiquitin ligases.
E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2 approximately ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.
16958690
A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B
Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. We have fractionated mitotic Xenopus egg extracts to identify components required for this process. We find that UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination. The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homologs of budding yeast CDC16 and CDC27. Because these proteins are required for anaphase in yeast and mammalian cells, we refer to this complex as the anaphase-promoting complex (APC). CDC27 antibodies deplete APC activity, while immunopurified CDC27 complexes are sufficient to complement either interphase extracts or a mixture of recombinant UBC4 and the ubiquitin-activating enzyme E1. These results suggest that APC functions as a regulated ubiquitin-protein ligase that targets cyclin B for destruction in mitosis.
834175
The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.
25281289
E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.
Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.
18951856
Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation
BACKGROUND
The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established.


RESULTS
We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4.


CONCLUSIONS
UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis.
3103893
Sequential E2s Drive Polyubiquitin Chain Assembly on APC Targets
The anaphase-promoting complex (APC), or cyclosome, is an E3 ubiquitin-protein ligase that collaborates with E2 ubiquitin-conjugating enzymes to assemble polyubiquitin chains on proteins important for cell-cycle progression. It remains unclear how the APC - or many other E3s - promotes the multiple distinct reactions necessary for chain assembly. We addressed this problem by analyzing APC interactions with different E2s. We screened all budding yeast E2s as APC coenzymes in vitro and found that two, Ubc4 and Ubc1, are the key E2 partners for the APC. These proteins display strikingly different but complementary enzymatic behaviors: Ubc4 supports the rapid monoubiquitination of multiple lysines on APC targets, while Ubc1 catalyzes K48-linked polyubiquitin chain assembly on preattached ubiquitins. Mitotic APC function is lost in yeast strains lacking both Ubc1 and Ubc4. E2-25K, a human homolog of Ubc1, also promotes APC-dependent chain extension on preattached ubiquitins. We propose that sequential E2 proteins catalyze K48-linked polyubiquitination and thus proteasomal destruction of APC targets.
16843994
UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit
The anaphase-promoting complex (APC/C), a ubiquitin ligase, is the target of the spindle-assembly checkpoint (SAC), and it ubiquitylates protein substrates whose degradation regulates progress through mitosis. The identity of the ubiquitin-conjugating (E2) enzymes that work with the APC/C is unclear. In an RNA interference (RNAi) screen for factors that modify release from drug-induced SAC activation, we identified the E2 enzyme UBE2S as an APC/C auxiliary factor that promotes mitotic exit. UBE2S is dispensable in a normal mitosis, but its depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage. In vitro, UBE2S elongates ubiquitin chains initiated by the E2 enzymes UBCH10 and UBCH5, enhancing the degradation of APC/C substrates by the proteasome. Indeed, following release from SAC-induced mitotic arrest, UBE2S-depleted cells neither degrade crucial APC/C substrates, nor silence this checkpoint, whereas bypassing the SAC through BUBR1 depletion or Aurora-B inhibition negates the requirement for UBE2S. Thus, UBE2S functions with the APC/C in a two-step mechanism to control substrate ubiquitylation that is essential for mitotic exit after prolonged SAC activation, providing a new model for APC/C function in human cells.
20172824
UBE2S drives elongation of K11-linked ubiquitin chains by the Anaphase-Promoting Complex
The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.
4918240
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.
4909737
Ubiquitin chain elongation requires E3-dependent tracking of the emerging conjugate.
Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.
8789627
APC/C-mediated multiple monoubiquitination provides an alternative degradation signal for cyclin B1
The anaphase-promoting complex or cyclosome (APC/C) initiates mitotic exit by ubiquitylating cell-cycle regulators such as cyclin B1 and securin. Lys 48-linked ubiquitin chains represent the canonical signal targeting proteins for degradation by the proteasome, but they are not required for the degradation of cyclin B1. Lys 11-linked ubiquitin chains have been implicated in degradation of APC/C substrates, but the Lys 11-chain-forming E2 UBE2S is not essential for mitotic exit, raising questions about the nature of the ubiquitin signal that targets APC/C substrates for degradation. Here we demonstrate that multiple monoubiquitylation of cyclin B1, catalysed by UBCH10 or UBC4/5, is sufficient to target cyclin B1 for destruction by the proteasome. When the number of ubiquitylatable lysines in cyclin B1 is restricted, Lys 11-linked ubiquitin polymers elaborated by UBE2S become increasingly important. We therefore explain how a substrate that contains multiple ubiquitin acceptor sites confers flexibility in the requirement for particular E2 enzymes in modulating the rate of ubiquitin-dependent proteolysis.
232961
Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis
Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.
11843543
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.
3554372
Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases.
Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.
4463635
Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome
The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B–Bmi1–UbcH5c E3–E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.
26081332
Structural assembly of cullin-RING ubiquitin ligase complexes.
The cullin-RING ubiquitin ligases (CRLs) are the largest family of multi-subunit E3 ligases in eukaryotes, which ubiquitinate protein substrates in numerous cellular pathways. CRLs share a common arched scaffold and a RING domain catalytic subunit, but use different adaptors and substrate receptors to assemble unique E3 machineries. In comparison to the first CRL structure, recent findings have revealed increased complexity in the overall architecture and assembly mode of CRLs, including multi-domain organization, inter-domain flexibility, and subunit dimerization. These features highlight the capacity of CRLs to catalyze protein ubiquitination under distinct cellular contexts and in response to diverse signals. As the first installment of a two-review series, this article will focus on recent advances in our understanding of CRL assembly mechanisms.
37414116
Structural regulation of cullin-RING ubiquitin ligase complexes.
Cullin-RING ligases (CRLs) compose the largest class of E3 ubiquitin ligases. CRLs are modular, multisubunit enzymes, comprising interchangeable substrate receptors dedicated to particular Cullin-RING catalytic cores. Recent structural studies have revealed numerous ways in which CRL E3 ligase activities are controlled, including multimodal E3 ligase activation by covalent attachment of the ubiquitin-like protein NEDD8, inhibition of CRL assembly/activity by CAND1, and several mechanisms of regulated substrate recruitment. These features highlight the potential for CRL activities to be tuned in responses to diverse cellular cues, and for modulating CRL functions through small-molecule agonists or antagonists. As the second installment of a two-review series, this article focuses on recent structural studies advancing our knowledge of how CRL activities are regulated.
17309741
APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex.
In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.
45227011
Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.
Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.
4450232
Molecular architecture and mechanism of the anaphase-promoting complex
The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C–substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C–coactivator–substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10–ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.
27193091
Insights into Degron Recognition by APC/C Coactivators from the Structure of an Acm1-Cdh1 Complex
Summary The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.
6938603
Features and development of Coot
Coot is a molecular-graphics program designed to assist in the building of protein and other macromolecular models. The current state of development and available features are presented.
4384228
Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex
SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-Å crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO–E2-thioester in an optimal orientation to enhance conjugation.
17715947
APC15 mediates CDC20 auto-ubiquitylation by APC/CMCC and MCC disassembly
The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/CMCC)-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.
46296632
Structural and Functional Analysis of the Human Mitotic-specific Ubiquitin-conjugating Enzyme, UbcH10*
Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-Å crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH2-terminal extension in UbcH10 is disordered and that a conserved 310-helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH2-terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.
6757737
Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation
Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-κB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2–Ubc13-ubiquitin (UEV–E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2–Ubc13-Ub complex to the acceptor-binding site of Mms2–Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.
12279623
Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI: http://dx.doi.org/10.7554/eLife.00828.001
34423567
Activation of UbcH5c~Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B.
Post-translational modification of proteins with ubiquitin is mediated by dynamic multienzyme machinery (E1, E2, and E3). E3 ubiquitin ligases play a key role acting as both scaffolds to bring reactants together and activators to catalyze ubiquitin (Ub) transfer from E2~Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2~Ub conjugate was found to affect the motions of E2~Ub and allosterically stimulate Ub transfer. This proposed mechanism implies that the dynamics of the conjugate, which has been shown to occupy a wide range of E2~Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2~Ub dynamics, we undertook an in-depth comparative analysis of (15)N nuclear magnetic resonance relaxation of UbcH5c~Ub in the absence and presence of the E3 ligase, E4B. Challenges encountered in deciphering interdomain motions for this ternary complex are discussed along with the limitations of the current approaches. Notably, although a reduction in interdomain dynamics of UbcH5c~Ub is observed upon binding to E4B, Ub retains an extensive degree of flexibility. These results provide strong support for our dynamic model of a significant orientational bias of Ub toward a more closed conformation in the E3/E2~Ub complex.
8295255
Regulation of ubiquitin chain initiation to control the timing of substrate degradation.
Processive reactions, such as transcription or translation, often proceed through distinct initiation and elongation phases. The processive formation of polymeric ubiquitin chains can accordingly be catalyzed by specialized initiating and elongating E2 enzymes, but the functional significance for this division of labor has remained unclear. Here, we have identified sequence motifs in several substrates of the anaphase-promoting complex (APC/C) that are required for efficient chain initiation by its E2 Ube2C. Differences in the quality and accessibility of these chain initiation motifs can determine the rate of a substrate's degradation without affecting its affinity for the APC/C, a mechanism used by the APC/C to control the timing of substrate proteolysis during the cell cycle. Based on our results, we propose that initiation motifs and their cognate E2s allow E3 enzymes to exert precise temporal control over substrate degradation.
39656042
A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination.
Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved alpha/beta-fold core domain of approximately 150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c approximately Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.
20875027
Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78.
The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.
205252033
E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6
In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process.
22420107
A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms.
Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the "backside" of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and RING-dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This demonstrates that an essential component of E3 complexes can simultaneously bind to E2 and enhance its loading with ubiquitin. These findings provide mechanistic insights into how ubiquitination can be stimulated.
5148780
A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment.
The Fizzy/Cdc20 family of proteins are essential activators of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. However, apart from the well-established role of the C-terminal WD40 domain in substrate recognition, the precise roles of the activators remain elusive. Here we show that Nek2A, which directly binds the APC/C, can be ubiquitylated and destroyed in Fizzy/Cdc20-depleted Xenopus egg extracts when only the N-terminal domain of Fizzy/Cdc20 (N-Cdc20) is added. This activity is dependent upon the C box and is conserved in the alternative activator, Fizzy-related/Cdh1. In contrast, canonical substrates such as cyclin B and securin require both the N-terminal and WD40 domains, unless N-Cdc20 is fused to substrates when the WD40 domain becomes dispensable. Furthermore, in Cdc20-depleted cells, N-Cdc20 can facilitate Nek2A destruction in a C box-dependent manner. Our results reveal a role for the N-terminal domain of the Fizzy/Cdc20 family of activators in triggering substrate ubiquitylation by the APC/C.
25057153
D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p.
The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation. We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation.
29815614
The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates.
Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 and Cdh1 leads to ubiquitin-dependent degradation of securin and cyclin B and thereby promotes the initiation of anaphase and exit from mitosis. Cyclin B and securin ubiquitination depend on a destruction box (D box) sequence in these proteins, but how APC/C bound to Cdc20 or Cdh1 recognizes the D box is poorly understood. By using site-specific photocrosslinking in combination with mutational analyses, we show that the D box directly interacts with an evolutionarily conserved surface on the predicted WD40 propeller structure of Cdh1 and that this interaction is essential for processive substrate ubiquitination. We further show that Cdh1 specifically crosslinks to the APC/C subunit Cdc27 and that Cdh1 binding to APC/C depends on the presence of Cdc27. Our data imply that APC/C is activated by the association of Cdh1 with Cdc27, which enables APC/C to recognize the D box of substrates via Cdh1's propeller domain.
36510169
Emi1 stably binds and inhibits the anaphase-promoting complex/cyclosome as a pseudosubstrate inhibitor.
The periodic destruction of mitotic cyclins is triggered by the activation of the anaphase-promoting complex/cyclosome (APC/C) in mitosis. Although the ability of the APC/C to recognize destruction box (D-box) substrates oscillates throughout the cell cycle, the mechanism regulating APC/C binding to D-box substrates remains unclear. Here, we show that the APC/C inhibitor Emi1 tightly binds both the APC/C and its Cdh1 activator, binds to the D-box receptor site on the APC/C(Cdh1), and competes with APC/C substrates for D-box binding. Emi1 itself contains a conserved C-terminal D-box, which provides APC/C-binding affinity, and a conserved zinc-binding region (ZBR), which antagonizes APC/C E3 ligase activity independent of tight APC binding. Mutation of the ZBR converts Emi1 into a D-box-dependent APC/C substrate. The identification of a direct Emi1-APC/C complex further explains how Emi1 functions as a stabilizing factor for cyclin accumulation and the need to destroy Emi1 for APC/C activation in mitosis. The combination of a degron/E3 recognition site and an anti-ligase function in Emi1 suggests a general model for how E3 substrates evolve to become pseudosubstrate inhibitors.
16194738
EM Structure of human APC/CCDH1-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/CCDH1 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/CCDH1 functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/CCDH1 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.
2061485
Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase promoting complex
The anaphase-promoting complex (APC) is the crucial ubiquitin ligase targeting the regulatory machinery of the cell cycle. Emi1, a major modulator of APC activity, is thought to act competitively as a pseudosubstrate. We show that the modulation of APC activity is more subtle: Emi1 inhibits ubiquitylation at both substrate binding and separately at the step of ubiquitin transfer to APC-bound substrates. The zinc-binding region of Emi1 allows multiple monoubiquitylation of substrates, but preferentially suppresses the ubiquitin chain elongation by UBCH10. Furthermore, the carboxy-terminal tail of Emi1 antagonizes chain elongation by Ube2S, by competitively preventing its binding to the APC cullin subunit through electrostatic interaction. Combinatorially, Emi1 effectively stabilizes APC substrates by suppressing ubiquitin chain extension. Deubiquitylating enzymes can then convert inhibited substrates to their basal state. Chain elongation may be a particularly sensitive step for controlling degradation, and this study provides the first kinetic evidence for how it is inhibited.
4370015
Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry
Oscillations in cyclin-dependent kinase (CDK) activity drive the somatic cell cycle. After entry into mitosis, CDKs activate the anaphase-promoting complex (APC), which then promotes cyclin degradation and mitotic exit. The re-accumulation of cyclin A causes the inactivation of APC and entry into S phase, but how cyclin A can accumulate in the presence of active APC has remained unclear. Here we show that, during G1, APC autonomously switches to a state permissive for cyclin A accumulation. Crucial to this transition is the APCCdh1-dependent autoubiquitination and proteasomal degradation of the ubiquitin-conjugating enzyme (E2) UbcH10. Because APC substrates inhibit the autoubiquitination of UbcH10, but not its E2 function, APC activity is maintained as long as G1 substrates are present. Thus, through UbcH10 degradation and cyclin A stabilization, APC autonomously downregulates its activity. This indicates that the core of the metazoan cell cycle could be described as a self-perpetuating but highly regulated oscillator composed of alternating CDK and APC activities.
18778672
The Processivity of Multiubiquitination by the APC Determines the Order of Substrate Degradation
The anaphase-promoting complex (APC) coordinates mitosis and G1 by sequentially promoting the degradation of key cell-cycle regulators. Following the degradation of its substrates in G1, the APC catalyzes the autoubiquitination of its E2 UbcH10. This stabilizes cyclin A and allows it to inactivate APC(Cdh1). How the APC establishes this complex temporal sequence of ubiquitinations, referred to as substrate ordering, is not understood. Here we show that substrate ordering depends on the relative processivity of substrate multiubiquitination by the APC. Processive substrates obtain ubiquitin chains in a single APC binding event. The multiubiquitination of distributive substrates requires multiple rounds of APC binding, which render it sensitive to lower APC concentrations, competition by processive substrates, and deubiquitination. Consequently, more processive substrates are preferentially multiubiquitinated in vitro and degraded earlier in vivo. The processivity of multiubiquitination is strongly influenced by the D box within the substrate, suggesting that substrate ordering is established by a mechanism intrinsic to APC and its substrates and similar to kinetic proofreading.
33305635
The unique N terminus of the UbcH10 E2 enzyme controls the threshold for APC activation and enhances checkpoint regulation of the APC.
In vitro, the anaphase-promoting complex (APC) E3 ligase functions with E2 ubiquitin-conjugating enzymes of the E2-C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2-C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2-E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N terminus increases substrate ubiquitination and the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N terminus to UbcH5 restricts ubiquitination activity but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2-E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.
19159205
Identification of conjugation specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2
Ubiquitin-like proteins (UBLs) modify targets via related E1-E2-E3 cascades. How is UBL conjugation fidelity established? Here we report the basis for UBL selection by UBL conjugating enzyme 12 (Ubc12), which is specific for the neural precursor cell expressed, developmentally down-regulated protein 8 (NEDD8), and does not form a thioester-linked conjugate with ubiquitin. We systematically identified Ubc12 surfaces impeding Ubc12∼ubiquitin conjugate formation and found that several structurally dispersed E1 binding elements, rather than UBL-interacting surfaces, determine E2∼UBL specificity. In addition to roles for conserved E1 and E2 domains, unique structures contribute UBL specificity to the NEDD8 and ubiquitin pathways. By removing surface elements, without substituting corresponding sequences from ubiquitin E2s, we unmasked Ubc12's vestigial preference for ubiquitin over NEDD8 by ∼1010-fold. This has implications for the evolution of specific functions among ubiquitin E2s. We also find that Ubc12 sequences dictating UBL selection map to the E3 binding site, thus providing a molecular mechanism preventing inappropriate modification of targets.
45091914
The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.
The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC.
16986330
Rapid E2-E3 Assembly and Disassembly Enable Processive Ubiquitylation of Cullin-RING Ubiquitin Ligase Substrates
Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic 'canyon' in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by crosslinking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved.
206214212
Structure of an APC3-APC16 complex: insights into assembly of the anaphase-promoting complex/cyclosome.
The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the "Platform" centers around a cullin-RING-like E3 ligase catalytic core; the "Arc Lamp" is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.