A comparative study of the cryo-EM structures of Saccharomyces cerevisiae and human anaphase-promoting complex/cyclosome (APC/C).

The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that controls progression through the cell cycle by orchestrating the timely proteolysis of mitotic cyclins and other cell cycle regulatory proteins. Although structures of multiple human APC/C complexes have been extensively studied over the past decade, the Saccharomyces cerevisiae APC/C has been less extensively investigated. Here, we describe medium resolution structures of three S. cerevisiae APC/C complexes: unphosphorylated apo-APC/C and the ternary APC/CCDH1-substrate complex, and phosphorylated apo-APC/C. Whereas the overall architectures of human and S. cerevisiae APC/C are conserved, as well as the mechanism of CDH1 inhibition by CDK-phosphorylation, specific variations exist, including striking differences in the mechanism of coactivator-mediated stimulation of E2 binding, and the activation of APC/CCDC20 by phosphorylation. In contrast to human APC/C in which coactivator induces a conformational change of the catalytic module APC2:APC11 to allow E2 binding, in S. cerevisiae apo-APC/C the catalytic module is already positioned to bind E2. Furthermore, we find no evidence of a phospho-regulatable auto-inhibitory segment of APC1, that in the unphosphorylated human APC/C, sterically blocks the CDC20C-box binding site of APC8. Thus, although the functions of APC/C are conserved from S. cerevisiae to humans, molecular details relating to their regulatory mechanisms differ.

Structure of the human outer kinetochore KMN network complex.

Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.

Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.

Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.

Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.

The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.

Molecular architecture of the augmin complex.

Accurate segregation of chromosomes during mitosis depends on the correct assembly of the mitotic spindle, a bipolar structure composed mainly of microtubules. The augmin complex, or homologous to augmin subunits (HAUS) complex, is an eight-subunit protein complex required for building robust mitotic spindles in metazoa. Augmin increases microtubule density within the spindle by recruiting the γ-tubulin ring complex (γ-TuRC) to pre-existing microtubules and nucleating branching microtubules. Here, we elucidate the molecular architecture of augmin by single particle cryo-electron microscopy (cryo-EM), computational methods, and crosslinking mass spectrometry (CLMS). Augmin's highly flexible structure contains a V-shaped head and a filamentous tail, with the head existing in either extended or contracted conformational states. Our work highlights how cryo-EM, complemented by computational advances and CLMS, can elucidate the structure of a challenging protein complex and provides insights into the function of augmin in mediating microtubule branching nucleation.

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

CDC20 is a coactivator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole-exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with ovarian germ cell tumors in two families. Functional characterization showed these mutants retain APC/C activation activity but have impaired binding to BUBR1, a component of the SAC. Expression of L151R and N331K variants promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. Generation of mice carrying the N331K variant using CRISPR-Cas9 showed that, although homozygous N331K mice were nonviable, heterozygotes displayed accelerated oncogenicity of Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor-promoting genes. SIGNIFICANCE: Two germline CDC20 missense variants that segregate with cancer in two families compromise the spindle assembly checkpoint and lead to aberrant mitotic progression, which could predispose cells to transformation. See related commentary by Villarroya-Beltri and Malumbres, p. 3432.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome.

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

Molecular mechanisms of APC/C release from spindle assembly checkpoint inhibition by APC/C SUMOylation.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls cell cycle transitions. Its regulation by the spindle assembly checkpoint (SAC) is coordinated with the attachment of sister chromatids to the mitotic spindle. APC/C SUMOylation on APC4 ensures timely anaphase onset and chromosome segregation. To understand the structural and functional consequences of APC/C SUMOylation, we reconstituted SUMOylated APC/C for electron cryo-microscopy and biochemical analyses. SUMOylation of the APC/C causes a substantial rearrangement of the WHB domain of APC/C's cullin subunit (APC2WHB). Although APC/CCdc20 SUMOylation results in a modest impact on normal APC/CCdc20 activity, repositioning APC2WHB reduces the affinity of APC/CCdc20 for the mitotic checkpoint complex (MCC), the effector of the SAC. This attenuates MCC-mediated suppression of APC/CCdc20 activity, allowing for more efficient ubiquitination of APC/CCdc20 substrates in the presence of the MCC. Thus, SUMOylation stimulates the reactivation of APC/CCdc20 when the SAC is silenced, contributing to timely anaphase onset.

Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP.

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

Crystal structure of the Cenp-HIKHead-TW sub-module of the inner kinetochore CCAN complex.

Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14-16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead-Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN-Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.

Structure of the DOCK2-ELMO1 complex provides insights into regulation of the auto-inhibited state.

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.

Architecture of the CBF3-centromere complex of the budding yeast kinetochore.

Kinetochores are multicomponent complexes responsible for coordinating the attachment of centromeric DNA to mitotic-spindle microtubules. The point centromeres of budding yeast are organized into three centromeric determining elements (CDEs), and are associated with the centromere-specific nucleosome Cse4. Deposition of Cse4 at CEN loci is dependent on the CBF3 complex that engages CDEIII to direct Cse4 nucleosomes to CDEII. To understand how CBF3 recognizes CDEIII and positions Cse4, we determined a cryo-EM structure of a CBF3-CEN complex. CBF3 interacts with CEN DNA as a head-to-head dimer that includes the whole of CDEIII and immediate 3' regions. Specific CEN-binding of CBF3 is mediated by a Cep3 subunit of one of the CBF3 protomers that forms major groove interactions with the conserved and essential CCG and TGT motifs of CDEIII. We propose a model for a CBF3-Cse4-CEN complex with implications for understanding CBF3-directed deposition of the Cse4 nucleosome at CEN loci.

Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution.

Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase-securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase-securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.

The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

Molecular basis of APC/C regulation by the spindle assembly checkpoint.

In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C­MCC complex (APC/C(MCC)). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20(MCC)) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced.

Molecular mechanism of APC/C activation by mitotic phosphorylation.

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.

The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells.

Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain.

Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5(N)). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5(N) has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5(N) shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.

Atomic structure of the APC/C and its mechanism of protein ubiquitination.

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.