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. Author manuscript; available in PMC: 2017 Aug 18.
Published in final edited form as: Mol Cell. 2016 Aug 10;63(4):593–607. doi: 10.1016/j.molcel.2016.07.003

Cryo EM of Mitotic Checkpoint Complex-bound APC/C reveals reciprocal and conformational regulation of ubiquitin ligation

Masaya Yamaguchi 1,6, Ryan VanderLinden 1,2,6, Florian Weissmann 3,6, Renping Qiao 3, Prakash Dube 4, Nicholas G Brown 1, David Haselbach 4, Wei Zhang 5, Sachdev S Sidhu 5, Jan-Michael Peters 3,*, Holger Stark 4,*, Brenda A Schulman 1,2,*
PMCID: PMC5148128  NIHMSID: NIHMS802991  PMID: 27522463

SUMMARY

The Mitotic Checkpoint Complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated Anaphase-promoting complex/Cyclosome (APC/CCDC20). APC/CCDC20 and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/CCDC20–UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here, electron microscopy reveals conformational variation of APC/CCDC20-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly, and conformational variability of APC/C, modulate UBE2C-catalyzed ubiquitylation of MCC’s CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/CCDC20–MCC modulating E2 activation underlies distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.

eTOC blurb

The Mitotic Checkpoint Complex (MCC) prevents APC/CCDC20-catalyzed ubiquitination of anaphase inhibitors until all chromosomes are properly bioriented. Cryo-EM and biochemistry reveal diverse conformations of APC/CCDC20-MCC modulating E2 activation and targeting, and suggest responsive mechanisms ensuring accurate chromosome segregation.

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INTRODUCTION

The massive, multisubunit E3 ligase Anaphase-promoting Complex/Cyclosome (APC/C) initiates chromosome segregation by directing ubiquitin (UB)-mediated proteolysis of anaphase inhibitors such as Securin, and other key mitotic regulators like cyclins. Regulation is achieved by post-translational modifications and the coordinated action of coactivators, E2 enzymes, and inhibitors (Sivakumar and Gorbsky, 2015). APC/C is activated in prophase by phosphorylation, which allows binding to the coactivator CDC20 (Fujimitsu et al., 2016; Qiao et al., 2016; Zhang et al., 2016). CDC20 binding enables substrate binding, and allosterically activates the catalytic core consisting of cullin (APC2) and RING (APC11) subunits (Burton et al., 2005; Chang et al., 2014; Kimata et al., 2008; Van Voorhis and Morgan, 2014; Zhang et al., 2016).

APC2 and APC11 are differentially harnessed by evolving ubiquitinated substrates and APC/C’s two partner E2s, UBE2C and UBE2S, for different forms of polyubiquitination (Brown et al., 2016), with UBE2C catalyzing UB ligation directly to substrates and UBE2S extending K11-linked chains. However, accurate cell division depends on APC/CCDC20 waiting until all chromosomes are properly bioriented on the spindle before triggering destruction of anaphase inhibitors (Jia et al., 2013; Primorac and Musacchio, 2013). Premature chromosome segregation and aneuploidy are averted by the spindle assembly checkpoint (SAC). The SAC monitors chromosome attachment and delays anaphase by producing the Mitotic Checkpoint Complex (MCC), which blocks APC/CCDC20-dependent ubiquitination of substrates such as Securin and cyclin B (Braunstein et al., 2007; Burton and Solomon, 2007; Fraschini et al., 2001; Hardwick et al., 2000; Herzog et al., 2009; Izawa and Pines, 2015; Kulukian et al., 2009; Sudakin et al., 2001).

Control of APC/CCDC20 activity in response to the status of the mitotic spindle involves tunable cycles of CDC20 synthesis and degradation, MCC assembly and disassembly, and APC/CCDC20-MCC association and dissociation (Foley and Kapoor, 2013; Foster and Morgan, 2012; Jia et al., 2013; Kim and Yu, 2011; Lara-Gonzalez et al., 2012; London and Biggins, 2014; Mansfeld et al., 2011; Musacchio, 2015; Musacchio and Ciliberto, 2012; Primorac and Musacchio, 2013; Uzunova et al., 2012). During chromosome alignment, APC/CCDC20 remains blocked by newly produced MCC replacing that which has dissociated. Checkpoint activation results in stabilization of MCC and APC/CCDC20-MCC complexes, whereas proper chromosome biorientation leads to liberation of APC/CCDC20, dismantling of MCC, and termination of MCC production.

A particularly vexing feature of checkpoint regulation is the reciprocal control of APC/CCDC20 and MCC (Reddy et al., 2007; Varetti et al., 2011). The evolutionarily conserved MCC “core” is a 3-protein complex consisting of a second molecule of CDC20, MAD2, and BUBR1/Mad3, although MCC from some organisms including humans also contains BUB3 (Izawa and Pines, 2015; Jia et al., 2013; Primorac and Musacchio, 2013). On the one hand, MCC acts as an inhibitor of APC/CCDC20 and blocks substrates. However, MCC appears to modulate activity of the priming E2, UBE2C, without impacting binding to the chain-forming E2 UBE2S (Kelly et al., 2014).

In the context of an APC/CCDC20–MCC complex, CDC20 can be ubiquitinated depending on the status of the checkpoint (Eytan et al., 2013; Ge et al., 2009; Jia et al., 2013; Jia et al., 2011; Mansfeld et al., 2011; Miniowitz-Shemtov et al., 2010; Musacchio, 2015; Musacchio and Ciliberto, 2012; Nilsson et al., 2008; Pan and Chen, 2004; Reddy et al., 2007; Uzunova et al., 2012; Varetti et al., 2011). Although CDC20 is a very short-lived protein, mechanisms and consequences of CDC20 ubiquitination remain under investigation. Ubiquitination may prevent accumulation of excess CDC20, potentially to avert mitotic slippage (Mansfeld et al., 2011; Nilsson et al., 2008). However, high UBE2C activity correlates with CDC20 ubiquitination and MCC-dissociation from APC/C, suggesting that UBE2C-mediated CDC20 ubiquitination contributes to checkpoint termination (Foster and Morgan, 2012; Reddy et al., 2007; Uzunova et al., 2012; Varetti et al., 2011). Accordingly, knocking down UBE2C and UBE2S decreased efficiency of release from the SAC and delayed progress from nuclear envelope breakdown to anaphase (Garnett et al., 2009; Jia et al., 2011; Kelly et al., 2014; Varetti et al., 2011; Wild et al., 2016; Williamson et al., 2009). Furthermore, CDC20 was stabilized and MCC accumulated on APC/C in checkpoint-arrested cells depleted for the APC15 subunit, or the corresponding Mnd2 in yeast (Foster and Morgan, 2012; Mansfeld et al., 2011; Uzunova et al., 2012). Notably, the only obvious catalytic defect of APC/C lacking APC15 (APC/CΔ15) is strikingly impaired CDC20 ubiquitination in the presence of MCC (Foster and Morgan, 2012; Uzunova et al., 2012).

Here we use electron microscopy (EM) and biochemistry to dissect regulation of APC/CCDC20 and MCC via ubiquitination by UBE2C and UBE2S. For clarity, we refer to the APC-bound activator CDC20 as CDC20A (i.e., in APC/CCDC20), and that in MCC as CDC20M. While MCC uniformly blocks substrate binding to APC/CCDC20, multiple conformations of APC/CCDC20–MCC, with rotation of the CDC20A–MCC assembly relative to APC/C and different positions for the APC2–APC11 catalytic core, determine if CDC20M is a substrate of UBE2C. Notably, the distribution of MCC complexes with APC/CCDC20 lacking APC15 (APC/CCDC20 Δ15) is shifted toward configurations that block UBE2C activation, while UBE2S binding and di-UB synthesis capabilities remain intact to presumably mediate rapid polyubiquitination of UB-primed CDC20. We propose that biasing APC/CCDC20–MCC conformation toggles ubiquitination of CDC20 as part of a dynamic response mechanism that can either sustain the SAC or trigger rapid onset of anaphase.

RESULTS

EM reconstructions reveal conformational variability of APC/CCDC20–MCC

We established recombinant systems for APC/CCDC20 (with glutamate replacements for 100 Ser/Thr sites of mitotic phosphorylation) that mimics the endogenous complex (Qiao et al., 2016; Weissmann et al., 2016), and for MCC by coexpressing its subunits in insect cells (Figure S1A–C). This allowed reconstituting MCC inhibition of UB ligation to fluorescent versions of Cyclin B’s N-terminal domain (CycBN*), Securin*, and Cyclin A* by APC/CCDC20, UBE2C, and UBE2S (Figure 1A).

Figure 1. Multiple APC/CCDC20–MCC conformations revealed by EM. See also Figure S1, Tables S1–S2, Movies S1–S3.

Figure 1

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(A) Ubiquitination of fluorescent CycBN*, Securin* and CycA*, by APC/CCDC20, titrating increasing concentrations of either full or core (CDC20M-BUBR1-MAD2) MCC.

(B) CryoEM reconstructions of representative APC/CCDC20-MCC CLOSED (left) and OPEN (right) conformations refined at 9Å and 10Å resolution, respectively (SDS-PAGE gel of APC/CCDC20-MCC, left).

(C) Superimposition of cryo EM maps for CLOSED and OPEN conformations of APC/CCDC20-MCC.

(D) Negative stain EM reconstructions of APC/CCDC20 in complex with full MCC, which contain BUB3.

(E) The overall CDC20A–MCC core assembly from docked crystal structures (Chao et al., 2012; Tian et al., 2012) is observed in both the CLOSED and OPEN configurations of APC/CCDC20-MCC.

(F) As in D, except with core MCC (CDC20M-BUBR1-MAD2) lacking BUB3.

(G) As in E, except with CDC20A–MCC core assembly docked negative stain EM maps from F.

(H) Negative stain EM reconstruction of endogenous MCC-bound APC/C (EMD-1591) (Herzog et al., 2009).

To gain structural insights into regulation, we analyzed APC/CCDC20-MCC by cryo electron microscopy (EM). Three-dimensional classification revealed several sub-populations with two globally distinctive configurations we term “CLOSED” and “OPEN”, which themselves are defined by multiple classes presumably reflecting their conformational dynamics. Cryo EM reconstructions for APC/CCDC20–MCC in the CLOSED and OPEN configurations were refined at resolutions of 9Å and 10Å, respectively (Table S1). Docking prior structures of human APC/CCDC20 and S. pombe MCC core (44% identical to human CDC20-BUBR1-MAD2) into the maps showed the arrangements of the two molecules of CDC20, with (A) bound to APC/C via activator binding sites and the other (M) in MCC (Chao et al., 2012; Izawa and Pines, 2015; Primorac and Musacchio, 2013; Zhang et al., 2016).

The distinct conformations display substantially different orientations of CDC20A–MCC with respect to APC/C (Figure 1B and 1C, Movies S1 and S2). In the “CLOSED” conformation, MCC fills the central cavity by engaging APC/CCDC20 on three sides. A continuous surface from CDC20M and BUBR1 engages CDC20A. BUBR1 interacts with the APC2–APC11 cullin–RING catalytic core. CDC20M contacts the available protomer (A) of the APC8 homodimer. When “OPEN”, the CDC20A–MCC assembly is preserved, but is localized away from the catalytic core.

Only the MCC core corresponding to CDC20-BUBR1-MAD2 was readily visible in the maps, despite the presence of BUB3 in the complex (Figure S1A). To validate this, we purified an MCC core complex that inhibited substrate ubiquitination as described (Izawa and Pines, 2015), with only slightly lower efficiency than full MCC in our assays (Figure 1A). Upon performing side-by-side negative stain EM analyses on APC/CCDC20 bound to full or core MCC, the resultant maps were superimposable, and there were not obvious major differences in the ratios of CLOSED and OPEN configurations among matched preps with or without BUB3 (Figure 1D–G, S1B–D). For comparison, retrospective inspection of the EM map from endogenous APC/CCDC20–MCC further validated that only the MCC core is apparent even in the presence of BUB3 (Figure 1H) (Herzog et al., 2009). This is consistent with conservation of key APC/CCDC20–MCC features across eukaryotes, as MCC lacks BUB3 in some organisms (Vanoosthuyse et al., 2009; Windecker et al., 2009). We speculate that BUB3 may be relatively flexibly tethered to APC/CCDC20–MCC, and thus all subsequent EM studies were performed with the MCC core complex unless otherwise stated (Table S1).

Reexamination of the published negative stain EM map of APC/C–MCC purified from checkpoint-arrested HeLa cells revealed that the dominant population (56%) corresponds to the CLOSED configuration, which may reflect higher propensity for purification due to additional contacts, or increased homogeneity of the CLOSED configuration, or relative stabilization of the CLOSED configuration during the checkpoint (Figure 1H) (Herzog et al., 2009). Blurring of the MCC moiety in the published map generated from unstained cryo EM data on endogenous APC/CCDC20–MCC may reflect conformational heterogeneity arising from the dynamic configurations (Herzog et al., 2009). Superimposing the EM maps corresponding to different 3D classes suggests a continuum of conformations where the CDC20A-MCC assembly rotates relative to APC/C. Notably, the APC2–APC11 catalytic core also exhibits conformational heterogeneity between the “CLOSED” and “OPEN” configurations, and among OPEN 3D classes even from a single sample (Movie S3).

MCC inhibition of substrate ubiquitination revealed by side-by-side comparison of APC/C, and APC/CCDC20 complexes with substrate or MCC

Cryo-EM reconstructions were aligned for phosphorylated apo APC/C (Figure 2A) (Zhang et al., 2016), substrate-bound APC/CCDC20 (Figure 2B) (Zhang et al., 2016), and CLOSED and OPEN conformations of APC/CCDC20-MCC (Figures 2C, 2D). Side-by-side comparison shows common mechanisms by which the CLOSED and OPEN configurations hijack substrate binding sites on APC/CCDC20, while differing in their interactions with the catalytic core.

Figure 2. “Snapshots” of distinct APC/C conformations associated with activation by CDC20 and modulation by MCC. See also Figure S2.

Figure 2

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(A) Prior apo phosphorylated APC/C (Zhang et al., 2016) showed the APC2 (green)-APC11 (blue) catalytic core as “DOWN”, blocking the canonical E2 binding site on APC11’s RING used by UBE2C. Cartoon depicts key elements related to MCC functions.

(B) Prior cryo EM map for APC/CCDC20–substrate complex (Zhang et al., 2016), showing substrate D-box (red) co-recruited to CDC20’s propeller (violet) and APC10 (pink). The catalytic core is activated by increased conformational mobility.

(C) Cryo EM map showing CLOSED configuration of APC/CCDC20–MCC. MCC blocks substrate-binding sites, and contacts APC2’s UBE2C-binding WHB domain and APC8 (protomer A).

(D) Cryo EM map showing OPEN configuration of APC/CCDC20–MCC. MCC blocks substrate-binding sites. APC/C’s central cavity is vacant and APC2–APC11 catalytic core is activated and less resolved, suggestive of mobiltity.

Prior structural data showed how CDC20 can allosterically activate E3 ligase activity. In the absence of CDC20, the canonical E2-binding site on APC11’s RING domain is masked, because the APC2–APC11 cullin-RING catalytic core occupies a “DOWN” position (Figure 2A) (Zhang et al., 2016). However, in the cryo EM maps for an APC/CCDC20-substrate complex, the APC2-APC11 catalytic core is relatively “UP” and freed from autoinhibition, and is visible only at lower contour and resolution presumably due to mobility (Figure 2B) (Zhang et al., 2016). This increases catalytic competence, because three domains of the catalytic core (the intermolecular cullin/RING “C/R” domain, the APC2 C-terminal “WHB” domain, and the APC11 C-terminal RING domain) are harnessed into different catalytic architectures for substrate ubiquitination by UBE2C and UB chain elongation by UBE2S (Brown et al., 2015; Brown et al., 2016; Chang et al., 2015).

In the CLOSED configuration, MCC captures the WHB domain from APC2, which is enabled by upward positioning of the C/R domain (Figure 2C). When APC/CCDC20–MCC is OPEN, the catalytic core is fully available, localized upward, and less visible, consistent with competence for binding to UBE2C (Figure 2D) (Brown et al., 2015; Brown et al., 2016; Chang et al., 2015).

In both configurations, MCC masks substrate-binding sites, by CDC20M and BUBR1 encasing the top-side of CDC20A’s β-propeller (Figures 1 and 2). Also, relative rotation of CDC20A’s β-propeller dismantles the D-box binding site between CDC20A and APC10 (Figure 2B–2D) (Herzog et al., 2009).

In separate sections below, we describe EM data (Table S1) and biochemistry defining elements mediating MCC inhibition of APC/CCDC20, mechanisms of CDC20 ubiquitination in the context of APC/CCDC20–MCC, and how APC/CCDC20–MCC structure and activity are modulated in the absence of APC15.

APC/CCDC20–MCC elements inhibiting substrate ubiquitination

The highest resolution Cryo-EM map obtained during the course of these studies was for the CLOSED configuration with an APC/CCDC20 mutant lacking APC15 (Figure S2A, Table S1, 4.8Å resolution by gold-standard Fourier Shell Correlation). Here we depict the CDC20A-MCC interface from this map, while effects of deleting APC15 are described in separate sections. Lower resolution for the CDC20A–MCC assembly of ≈6.5 Å presumably reflects relative mobility, but it was sufficient to dock prior high-resolution structures of MCC subunits for visualizing elements mimicking substrate D-, KEN-, and ABBA/Phe-box motifs (Figure 3, S2, S3).

Figure 3. Multiple related elements mediate substrate and MCC association with CDC20A. See also Figure S3.

Figure 3

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(A) Scheme of BUBR1 and CDC20 motifs.

(B) Close-up views of coactivator–D/CRY-box interactions from crystal structure CDH1–D-box (He et al., 2013) and cryo EM map (APC/CCDC20Δ15–MCC, CLOSED, 4.8 Å resolution) showing density for CDC20M CRY-box and D-box receptor region of CDC20A.

(C) Close-up views of CDC20-KEN-box interactions from crystal structure (Tian et al., 2012) and cryo EM map (APC/CCDC20Δ15–MCC core, CLOSED configuration, 4.8 Å resolution).

(D) Mutations in key MCC elements impair inhibition, detected by titrating increasing concentrations of MCC into reactions monitoring APC/CCDC20–mediated ubiquitination of the fluorescent substrate CycBN*.

(E) Mutations in CDC20A KEN-box binding site impair MCC-mediated inhibition, detected by reactions as in D. Note that CycBN* has a D-box but not a KEN-box, so its ubiquitination in absence of MCC is not substantially impaired by CDC20A KEN-box receptor mutant.

On one edge of CDC20A, there is potentially weak density in the D-box binding site, and stronger density for a Cys-Arg-Tyr (CRY) box immediately preceding CDC20M’s propeller domain (Figure 3A, 3B). The other edge of CDC20A apparently binds an ABBA/Phe-box-like motif (Di Fiore et al., 2015; Diaz-Martinez et al., 2015), which continues into a “pre-KEN sequence” and ultimately a KEN-box that are sandwiched between the CDC20A propeller and the CDC20M/BUBR1 surface from MCC (Figure 3A, 3C, S3A). Indeed, prior studies had identified D1- and KEN2 motifs in BUBR1, and the corresponding D- and KEN-box receptor sites on CDC20A as important for MCC binding to APC/C (Izawa and Pines, 2015). We further validated the cryo-EM data by testing effects of mutating BUBR1’s D1, ABBA-like, pre-KEN, and KEN2 motifs and CDC20M’s CRY box in MCC, as well as the KEN-receptor in CDC20A. All these structure-based mutations thwarted MCC inhibition of substrate ubiquitination (Figure 3D, 3E, S3B).

APC/CCDC20–MCC in the OPEN configuration is structurally poised for UBE2C-dependent ubiquitination of CDC20M

The two APC/CCDC20–MCC conformations present an intriguing dichotomy for the catalytic core. In particular, with the CDC20A-MCC assembly swung OPEN, APC/C’s central cavity is vacant and in principle could accommodate UBE2C (Figure 2D) (Brown et al., 2015; Chang et al., 2015). Although previous studies monitoring cellular CDC20 ubiquitination during the spindle assembly checkpoint could not distinguish between CDC20A and CDC20M (Foster and Morgan, 2012; Mansfeld et al., 2011; Uzunova et al., 2012), we used methylated UB that cannot form chains to directly probe targeting. CDC20A was distinguished by an N-terminal Myc-tag, and CDC20M by a FLAG-tag (Figure 4A). Adding MCC to APC/CCDC20 and the E2 UBE2C resulted in CDC20M ubiquitination and decreased modification of CDC20A (Figure 4B). CDC20M ubiquitination was decreased by mutating a pair of known CDC20 ubiquitination sites (Lys485 and 490) within MCC (Figure 4C) ((Mansfeld et al., 2011) and www.phosphosite.org).

Figure 4.

Figure 4

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APC/CCDC20–MCC OPEN configuration directs UBE2C-catalyzed ubiquitination of CDC20M. See also Figure S4

(A) Scheme of 2-color western blot distinguishing CDC20A and CDC20M in ubiquitination assays.

(B) APC/CCDC20 and UBE2C-dependent modification of CDC20 monitored in reactions with methyl-UB that can be linked to substrate but cannot form chains.

(C) Reactions as in B, showing roles of known CDC20 ubiquitination sites, by comparing APC/CCDC20 and UBE2C-dependent ubiquitination for WT and K485R/K490R CDC20M.

(D) Scheme for purifying APC/CCDC20-UBE2C-MCC core, with CDC20M residue 490 (corresponding to ubiquitination site) cross-linked to active site of UBE2C.

(E) Negative stain EM reconstruction of APC/CCDC20-UBE2C-MCC core representing CDC20M ubiquitination, with CDC20A-MCC rotated in the OPEN configuration. Inset: close-up of model showing key elements to recruit/activate/position UBE2C from prior crystal structures that is docked in EM map (Brown et al., 2015; Brown et al., 2014).

(F) Assay as in B testing cullin (APC2) and RING (APC11) architecture in recruiting/positioning/activating UBE2C for UB ligation to CDC20M, through deleting UBE2C-binding (APC2 WHB) and activating (APC11 RING) domains from APC/C, or mutating corresponding contact residues from UBE2C (F53 and Y91, respectively) (Brown et al., 2014).

To visualize the APC/CCDC20–MCC–UBE2C architecture poised for CDC20M ubiquitination, we modified our published approach to capture targeting by UBE2C (Brown et al., 2015; Brown et al., 2016). Briefly, the homobifunctional crosslinker BMOE joined UBE2C’s active site Cys with a Cys corresponding to Lys490 in a modified CDC20M (Figure 4D). A purified BMOE-linked MCC–UBE2C complex was assembled with APC/CCDC20 for structural analysis by negative stain EM (Figure 4D, 4E). The EM reconstruction shows the OPEN configuration. Docking prior crystal structures shows how APC2’s WHB and APC11’s RING domains co-engage UBE2C (Brown et al., 2015; Brown et al., 2014). This places UBE2C in a parallel location as poised for substrate ubiquitination, except UBE2C’s active site is juxtaposed with CDC20M (Figure 4E, S4) (Brown et al., 2015; Chang et al., 2015). We mutationally tested the mechanism, removing APC2’s UBE2C-binding WHB domain or APC11’s UBE2C-activating RING domain from APC/CCDC20, or with UBE2C F53D and Y91D point mutations in the APC2- and APC11-binding sites respectively (Brown et al., 2015; Brown et al., 2014). The mutants eliminated CDC20M ubiquitination, suggesting a crucial role for the APC2-APC11 cullin-RING mechanism activating UBE2C~UB (Figure 4F, S4) (Brown et al., 2015).

Cryo EM Maps of APC/CCDC20–MCC without the APC15 Subunit Show a Shift toward Conformations Inhibiting the Activation of UBE2C

To understand how APC/CCDC20–MCC can be regulated, we considered that depleting the APC/C subunit APC15 selectively impairs CDC20 ubiquitination during checkpoint regulation (Foster and Morgan, 2012; Mansfeld et al., 2011; Uzunova et al., 2012). We discovered this is an intrinsic property of APC/C, because in comparison to the WT complex, recombinant APC/CCDC20 prepared without APC15 (APC/CCDC20Δ15) retained substrate ubiquitination activity that was inhibited by MCC, but MCC ubiquitination is selectively impaired (Figure 5A, 5B, S5A).

Figure 5. Deletion of APC15 influences APC/CCDC20–MCC conformational regulation and activity with the E2 UBE2C. See also Figure S5, Table S2, Table S3.

Figure 5

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(A) Western blot (anti- APC3, APC10, and APC15) confirming lack of APC15 in recombinant APC/CCDC20Δ15, compared to WT control.

(B) Recombinant APC/CCDC20Δ15 recapitulates key endogenous properties. Left, summary from (Foster and Morgan, 2012; Mansfeld et al., 2011; Uzunova et al., 2012). Right, deleting APC15 from recombinant APC/CCDC20 has no obvious impact on UBE2C-dependent substrate ubiquitination in absence of MCC but impairs ubiquitination of CDC20M from MCC, tested by simultaneous detection of fluorescent CycBN* substrate and FLAG-CDC20M in reaction.

(C) Cryo EM map of APC/CCDC20Δ15 complex representing substrate ubiquitination with UBE2C (UBE2C active site crosslinked to substrate, 6.1 Å resolution, SDS-PAGE gel, left). Cartoon of catalytic architecture for substrate ubiquitination is shown below.

(D) Cryo EM map of APC/CCDC20Δ15-MCC in CLOSED configuration (4.8 Å resolution) resolving elements blocking substrate binding and inhibiting APC/C catalytic core in the UP (activated) position.

(E) Cryo EM map showing APC/CCDC20Δ15-MCC in distinctive OPEN configuration (9 Å resolution) with catalytic core in inactive DOWN position. The UBE2C-binding site on APC11 RING domain is blocked with APC2-APC11 in DOWN position previously described for apo-APC/C (Chang et al., 2014; Zhang et al., 2016).

(F) Side-by-side pairwise comparison of density corresponding to APC2–APC11 catalytic core after superimposing cryo EM maps for apo APC/C and APC/CCDC20 (Zhang et al., 2016) and APC/CCDC20Δ15–MCC in the atypical inactive OPEN configuration shown in E. CDC20A-binding to apo APC/C typically induces repositioning of the APC2–APC11 catalytic core from a “DOWN” position to an “UP” orientation, which enables UBE2C to bind APC2’s WHB and APC11’s RING domains (Brown et al., 2015; Chang et al., 2015; Zhang et al., 2016). The minor population of APC/CCDC20Δ15–MCC in the OPEN configuration is inactive due to the catalytic core occupying the DOWN orientation.

(G) The ratio of CDC20A–MCC populations in CLOSED versus OPEN configurations observed in negative stain EM reconstructions of 7 matched purifications of MCC bound to WT or Δ15 APC/CCDC20 (Table S2). Samples with no OPEN classes are denoted (≈).

(H) Among the classes in OPEN configurations analyzed in G, the poplulations with the APC2–APC11 catalytic core in activated UP versus inactive DOWN positions were compared for WT or Δ15 APC/CCDC20 (Table S3).

(I) Comparing effects of MCC on WT or APC/CCDC20Δ15-stimulated activation of UBE2C~UB, as monitored by hydrolysis of oxyester-linked UBE2C (catalytic Cys-to-Ser)~UB into free UBE2C and UB.

(J) Comparing MCC inhibition of WT or APC/CCDC20Δ15-stimulated activation of UBE2C~UB by quantification of UBE2C~UB remaining after 3 hours from experiments as in I. Error = SEM, n=3.

To compare structural effects of deleting APC15 on substrate ubiquitination versus on interactions with MCC, we first adapted our method for visualizing an assembly mimicking an APC/CCDH1-UBE2C~UB-substrate intermediate (Brown et al., 2015). We determined a cryo EM reconstruction of APC/CCDC20Δ15 bound to a 3-way crosslinked complex linking the preferred ubiquitination site, UBE2C, and a donor UB (6.1 Å resolution, gold-standard Fourier Shell Correlation, Figure 5C, S5B). In the maps, the most static regions extend to ≈4 Å in resolution, with lower resolution for catalytic core presumably arising from dynamic properties required for ubiquitination, The map resembled that representing an APC/CCDH1-UBE2C~UB-substrate intermediate, with UBE2C-bound to APC2–APC11 adjacent to substrate, and with the donor UB not visible presumably due to conformational flexibility (Figure S5C, S5D) (Brown et al., 2015; Brown et al., 2014; Zhang et al., 2016). The most obvious effects of deleting APC15 were lack of APC15, and disappearance of density for and rearrangement of APC15-binding helices from APC5 (Figure S5E).

We analyzed conformations of APC/CCDC20Δ15-MCC by cryo EM to understand how ubiquitination is impaired. Maps corresponding to CLOSED and OPEN configurations were refined at overall resolutions of 4.8Å and 9Å respectively (Figure 5D, 5E, S2A). In comparison to the WT complex, the complex lacking APC15 showed an obvious increase in the population of the CDC20M-MCC assembly in the CLOSED configuration, accompanied by relative rotation in APC4/APC5 regions (Figure S5E). This would block binding to UBE2C due to BUBR1 hijacking the WHB domain from APC2 (Figure 5D). UBE2C activation is also blocked in the less populated class with MCC in the OPEN orientation, because APC2-APC11 was in the DOWN position typically observed in apo-APC/C, which occludes UBE2C binding to the RING (Figure 2A, 5E, 5F).

We tested if deleting APC15 indeed would hinder APC/CCDC20–MCC-dependent activation of UBE2C. First, we obtained structural data by collecting negative stain EM datasets, calculating maps, and quantifying populations in different conformations for 14 samples from 7 matched preps with or without APC15. In the preps lacking APC15, the proportion forming the CLOSED configuration was relatively increased, while those classes with the OPEN CDC20A-MCC configuration predominantly displayed the catalytic core in the DOWN orientation blocked for UBE2C-binding (Figure 5G, 5H; Table S2, S3). Second, we directly tested effects of deleting APC15 and/or adding MCC on activation of UBE2C~UB, by monitoring hydrolysis of an oxyester-bonded UBE2C~UB conjugate with UB linked to Ser replacing UBE2C’s catalytic Cys. In agreement with the structural data, adding APC/CCDC20 stimulates hydrolysis of the oxyester-bonded UBE2C~UB conjugate irrespective of the presence or absence of APC15, while deleting APC15 enhanced inhibition by MCC (Figure 5I, 5J).

Although activity was greatly reduced, low-level hydrolysis of UBE2C~UB (Figure 5I, 5J) raised the possibility that APC15 is not absolutely required for APC/CCDC20–MCC to adopt a conformation that activates UBE2C~UB. This may be enhanced or more detectable at high protein concentrations such as used in the hydrolysis assay. To test if APC/CCDC20Δ15–MCC can indeed attain an active conformation with UBE2C, we assembled the crosslinked MCC–UBE2C complex with APC/CCDC20Δ15 for structural analysis by negative stain EM (Figure 6A). The map was strikingly similar to that from the corresponding complex with WT APC/CCDC20 (Figure 4E). Overall, the data suggest that APC/CCDC20Δ15–MCC can in principle activate UBE2C~UB, but that the active conformation is not preferred.

Figure 6. Elements distinctly mediating MCC interactions with APC/C subunits in CLOSED configuration determine inhibition of CDC20M ubiquitination. See also Figure S5.

Figure 6

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(A) APC15 is not absolutely required for APC/CCDC20-UBE2C to adopt catalytic architecture for CDC20M ubiquitination. Negative stain EM reconstruction of APC/CCDC20Δ15-UBE2C-MCC core shows that forcing juxtaposition of UBE2C and CDC20M by crosslinking enables visualizing catalytic assembly as in Figure 4E.

(B) Close-up of BUBR1–APC2 WHB domain interactions distinctive for the CLOSED configuration of APC/CCDC20–MCC, with crystal structures (Bolanos-Garcia et al., 2011; Brown et al., 2015) docked in cryo EM map from APC/CCDC20Δ15-MCC and showing BUBR1 residues mutated in E and I.

(C) Crystal structure of APC2 WHB domain bound to UBE2C (Brown et al., 2015) oriented as APC2 WHB-domain–BUBR1 interface in B shows how MCC in CLOSED configuration blocks UBE2C binding.

(D) Close-up of APC8 (A) TPR pocket showing density attributed to CDC20M IR tail, an interaction unique to the CLOSED configuration of APC/CCDC20–MCC.

(E) Mutating APC2-binding residues from BUBR1 substantially restores APC/CCDC20Δ15-stimulated hydrolysis of oxyester-linked UBE2C~UB.

(F) Comparing APC/CCDC20Δ15-stimulated activation of UBE2C~UB in presence of WT or BUBR1 mutant MCC by quantification of UBE2C~UB remaining after 3 hours from experiments as in E. Error = SEM, n=3.

(G) Mutating IR tail from CDC20M in MCC, but not a negative control region (C-box), substantially restores APC/CCDC20Δ15-stimulated hydrolysis of oxyester-linked UBE2C~UB.

(H) Quantification of UBE2C~UB after 3 hours from experiments as in G. Error = SEM, n=3.

(I) Mutating APC2-binding residues from BUBR1 restores CDC20M ubiquitination by APC/CCDC20Δ15 and UBE2C.

(J) Mutating CDC20M IR-tail restores ubiquitination by APC/CCDC20Δ15 and UBE2C.

Since the EM maps of APC/CCDC20Δ15-MCC in the CLOSED configuration uniformly showed APC2-APC11 in the activated UP location but with UBE2C access blocked by MCC, we hypothesized that mutations disrupting key interactions unique to the CLOSED configuration should enable activation of UBE2C~UB and ubiquitination of CDC20M even by APC/CCDC20Δ15. One interaction unique to the CLOSED configuration involves MCC hijacking the UBE2C-binding surface of APC2’s WHB domain (Figure 6A–C). Another involves peptide-like density terminating in the available tetratricopeptide repeat (TPR) groove from APC8 (protomer A of the APC8 homodimer) (Figure 6D). We attributed this density to CDC20M’s IR-tail, due to strikingly similarity to Ile-Arg (IR) tails from APC10, CDH1, and CDC20A bound to TPR groves from the two APC3 protomers (Figure S5F) (Chang et al., 2015; Zhang et al., 2016). Also, APC8A is in principle competent to bind an Ile and Arg, as protomer B contacts such residues within a coactivator’s C-box motif (Chang et al., 2015; Zhang et al., 2016) (Figure S5F), while APC8A is poised to bind CDC20M’s IR-tail with MCC positioned in the CLOSED configuration. Indeed, structure-based mutations in the BUBR1 surface that binds the APC2 WHB domain, or in CDC20M’s IR-tail, substantially restored the ability of APC/CCDC20Δ15 to hydrolyze UBE2C~UB in the presence of MCC (Figure 6E–H), and to catalyze CDC20M ubiquitination (Figure 6I, 6J).

UBE2S-dependent UB-chain synthesis in the presence of MCC

To understand how MCC binding would affect UB chain formation, we inspected the cryo-EM maps for potential to form the specialized APC/C architecture juxtaposing UBE2S’s active site with an acceptor UB. A prior cryo-EM reconstruction, obtained by crosslinking UBE2S’s catalytic Cys to a substrate-fused UB variant (UBv) with enhanced affinity for the acceptor UB-binding site, indicated that UB chain elongation by UBE2S involves a completely different catalytic architecture from that activating UBE2C (Brown et al., 2016). Instead, UBE2S’s C-terminal peptide (CTP) binds an APC2/APC4 groove, while UBE2S’s catalytic domain contacts a pair of APC2 C/R domain helices. The unique interactions direct UBE2S’s active site toward an acceptor UB recruited to a distinct APC11 RING surface (Brown et al., 2016; Brown et al., 2014; Kelly et al., 2014). Notably, prior data showed that MCC did not inhibit UBE2S-dependent synthesis of unanchored di-UB chains with endogenous APC/CCDC20 (Kelly et al., 2014).

Even in the CLOSED configuration of APC/CCDC20–MCC, the UBE2S and acceptor UB binding sites appeared available (Figure 7A, S6A)(Kelly et al., 2014). To test if UBE2S activity is spared with MCC predominantly bound in the CLOSED configuration, we examined UBE2S-dependent UB transfer to a fluorescein-labeled acceptor UB*, and we saw no defect from deleting APC15 (Figure 7B). To visualize this, we adapted our published approach and performed cryo EM on APC/CCDC20Δ15–MCC bound to a complex with UBE2S’s catalytic Cys crosslinked to the UBv as a proxy for an acceptor UB (Brown et al., 2016) (Figure 7C). A map for the CLOSED configuration was refined at 5.7Å resolution, which allowed placement of APC/CCDC20Δ15–MCC, while the local resolution for the catalytic core was ≈10Å (Figure 7D, S6A–B Table S1). The position of UBE2S–UBv was approximated based on prior cryo-EM data, where UBE2S was more avidly anchored with the Ubv fused to a substrate (Figure 7D, S6A) (Brown et al., 2016).

Figure 7. Multiple catalytic and conformational mechanisms contributing to reciprocal regulation of APC/CCDC20 and MCC. See also Figure S6, S7.

Figure 7

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(A) Left, distinctive catalytic architecture for UBE2S-mediated di-UB synthesis (Brown et al., 2016). Distinct surface of APC11 RING recruits acceptor UB. APC2 C/R domain activates UBE2S catalytic domain. APC2/APC4 (propeller domain, PD) groove recruits UBE2S’s unique C-terminal peptide (CTP). Key elements (APC11 RING UB-binding site, APC2–APC11 C/R domain, and APC2/APC4 groove) required for UB chain synthesis by UBE2S are available in the presence of MCC.

(B) Reactions with or without MCC monitoring WT or Δ15 APC/CCDC20 and UBE2S-catalyzed substrate-independent di-UB* synthesis. UBE2S control mutants (I154A and L222A) confirm role of catalytic architecture (Brown et al., 2016; Brown et al., 2014; Kelly et al., 2014).

(C) SDS-PAGE gel of purified APC/CCDC20Δ15 complex with MCC core and 3-way UBE2S-UBv-UB complex to represent UB chain elongation. UBv is a UB variant with enhanced affinity for the acceptor UB binding surface of APC11 RING (Brown et al., 2016).

(D) Cryo EM map of complex from C, representing overall conformational compatibility for UBE2S-dependent UB-chain formation with APC/CCDC20Δ15–MCC in the CLOSED configuration.

(E) Experiment testing if UBE2S in presence of UBE2C catalyzes UB chain elongation on MCC’s CDC20M, with WT APC/CCDC20 or the mutant lacking APC15.

(F) Summary of B–E, Figure S6C–D. MCC inhibits D/KEN recruitment but not intrinsic chain forming activity of APC/CCDC20–UBE2S irrespective of APC15-dependent conformational modulation.

(G) Conformations of APC/CCDC20–MCC determine reciprocal regulation. MCC degron-like elements mediate binding to CDC20A to inhibit substrate ubiquitination (i, ii). Rotation of the CDC20A-MCC assembly and APC/C conformation determine if UBE2C is blocked from the APC2–APC11 catalytic core (iii, iv) or if is CDC20 can be ubiquitinated by UBE2C (v). After an initial UB is linked, a UB chain can be extended by UBE2S.

The structural data confirmed that MCC blocks substrate binding sites on CDC20A, while allowing UBE2S placement adjacent to the UB-binding site on APC11’s RING domain (Figure 7D). This led to several predictions. First, UBE2S should extend polyUB chains initiated by UBE2C and APC/CCDC20–MCC. We confirmed this for CDC20M, as higher molecular weight conjugates were observed upon adding UBE2S, but there was no modification by APC/CCDC20Δ15 that inhibited UBE2C-dependent priming of CDC20M (Figure 7E, S6C). Second, if a substrate is already marked by a UB, such as a UB-Securin* fusion, then the level of UBE2S-dependent chain growth would reflect that occurring upon MCC inhibition of D- and KEN-box access to CDC20A irrespective of whether APC/CCDC20-MCC was CLOSED or OPEN. Indeed, the products of APC/CCDC20–UBE2S-catalyzed ubiquitination reactions were similar with UB-Securin* in the presence of MCC or a different competitor (Hsl1) blocking the D- and KEN-box sites on CDC20A, or in the absence of MCC but with a D- and KEN-box mutant UB-Securin* (Figure S6D, S6E). Thus, MCC competes with substrate binding, but if an acceptor UB is accessible, UBE2S can elongate chains even with CDC20A-MCC in the closed configuration (Figure 7F).

DISCUSSION

Overall, our data define conformational control and reciprocal regulation of APC/CCDC20 and MCC (Figure 7G). First, the cryo EM data reveal elements blocking degron motifs from accessing APC/CCDC20-MCC explaining how substrate ubiquitination is inhibited (Figure 3, S3). Second, a range of CDC20A-MCC orientations, coupled with conformational dynamics of the APC/C catalytic core, endows APC/CCDC20–MCC with variable functions. Although targeting with UBE2C is blocked when APC/CCDC20–MCC is CLOSED, the OPEN configuration places CDC20M ubiquitination sites (Lys485 and Lys490) for modification by activated UBE2C (Figure 4E). Since MCC does not impair UBE2S association (Kelly et al., 2014), polyubiquitination could occur rapidly in cells after CDC20M is initially primed by UBE2C (Figure 7E).

At this point, how APC/CCDC20–MCC conformations are naturally toggled remains poorly understood. The strong stabilization of APC/C-MCC upon checkpoint activation in cells depleted of APC15 (Foster and Morgan, 2012; Mansfeld et al., 2011; Uzunova et al., 2012) raises the possibility that activating the checkpoint may involve shifting APC/CCDC20–MCC to CLOSED or DOWN configurations. By contrast, mechanisms opening APC/CCDC20-MCC would increase UBE2C~UB activation and ubiquitination of CDC20M, much like our mutations removing contacts unique to the CLOSED conformation (Figure 6). It seems that UBE2C access is a crucial determinant controlling the reciprocal regulation of APC/CCDC20 and MCC. Indeed, UBE2C is a mediator of checkpoint silencing (Jia et al., 2011; Reddy et al., 2007; Varetti et al., 2011). UBE2C could potentially act as a wedge capturing APC/CCDC20–MCC as it swings open. APC/CCDC20–MCC conformation could also be influenced by factors affecting the APC4–APC5 region, which resembles a lever arm poised as a conduit between CDC20A-MCC and the catalytic core (Figure S5E). This could potentially be regulated by several phosphorylation sites in APC5, which are substituted by glutamates in our recombinant APC/CCDC20 (Qiao et al., 2016).

BUB3 also regulates termination of the checkpoint (Vanoosthuyse et al., 2009; Windecker et al., 2009), but is not visible in the EM maps (Figure 1). Interestingly, BUB3-mediated checkpoint silencing may involve binding to “M-E-L-phosphoT” sequences, and we note that such a sequence invisible in APC5 in the cryo-EM maps is potentially positioned to modulate APC/CCDC20–MCC conformation (Vleugel et al., 2015; Yamagishi et al., 2012). The overall similarity of EM maps for APC/CCDC20–MCC complexes with or without BUB3 also raises the question as to whether UBE2C could ubiquitinate CDC20 in the context of assembly/disassembly intermediates containing only subsets of MCC components (Eytan et al., 2013; Han et al., 2013).

It seems likely that the configurations of APC/CCDC20–MCC mediating various roles (inhibition, CDC20M ubiquitination, or other functions such as MCC dissociation) rely on multisite interactions, where each element is weak on its own, but synergistic formation or dismantling of multiple contacts governs conformational control of distinctive activities. Multisite regulation is emerging as a common mechanism controlling APC/C activities, including EMI1 inhibition of interphase APC/CCDH1 (Chang et al., 2015; Frye et al., 2013; Miller et al., 2006; Wang and Kirschner, 2013), and recruitment, positioning, and activation of APC/C’s partner E2s to achieve different forms of ubiquitination (Brown et al., 2015; Brown et al., 2016; Brown et al., 2014; Chang et al., 2015; Kelly et al., 2014). Multisite interactions can also allow E3 regulators to straddle multiple functions (Figure S7). MCC represents a distinct manifestation of E3 modulator, as pan-inhibitor or selective substrate of APC/CCDC20 as needed (Figure 7G). It seems likely that there will be many other cases where subtle variations in regulation can determine when a single E3 regulator is an inhibitor, substrate, or modulator of ubiquitin ligation to establish dynamic cellular regulation.

EXPERIMENTAL PROCEDURES

Proteins and assays

Proteins used in this study are human except for yeast Hsl1. Recombinant APC/C and its variants contain 100 phosphomimetic mutations (Qiao et al., 2016; Weissmann et al., 2016). CDC20, MCC and variants were expressed in Hi5 insect cells and then purified by nickel affinity, cation exchange and size exclusion chromatography.

Substrate ubiquitination assays were performed as described, with MCC titrated from 15–250 nM (Brown et al., 2016; Qiao et al., 2016). CDC20 ubiquitination assays were adapted from (Foe et al., 2011; Foster and Morgan, 2012) to use methyl UB, and extended to include UBE2S with WT UB. To distinguish CDC20 ubiquitination targets, CDC20A and CDC20M were N-terminally Myc- and Flag-tagged, respectively, for 2-color western detection by secondary antibodies conjugated with DyLight 488 and Alexa 633, respectively, during a single scan with Typhoon FLA 9500.

Hydrolysis of oxyester-linked UBE2C~UB (Brown et al., 2015) was performed three independent times, with 1 μM APC/C or APC/CΔ15, 1 μM CDC20, 5 μM UBE2C~UB, ± 1 μM MCC at 30°C. Reaction products were visualized in Coomassie blue-stained 4–12% NuPAGE gels (Life Technologies).

Complex preparation for EM

Complexes were purified by sequential affinity pulldowns, followed by specific polishing steps. APC/CCDC20, and APC/CCDC20-MCC, or versions lacking APC15, were prepared by either coexpressing APC/C, CDC20, and/or MCC, or by mixing lysates from Hi5 insect cell cultures expressing components independently. Subcomplexes crosslinked to E2 active sites [UBE2C-substrate-UB (FLAG-tagged donor UB mimic), UBE2C-MCC (His6-FLAG-tagged BUBR1), UBE2S-UBv-UB (untagged)] were generated largely as described previously (Brown et al., 2015; Brown et al., 2016). 2- or 3-way crosslinking between cysteine side-chains was performed through use of BMOE or TMEA (Pierce), respectively. Complexes were initially purified based on affinity tag(s) on APC/C, either a C-terminal Twin-Strep tag on APC4 or sequentially via an N-terminal Twin-Strep tag on APC2 and N-terminal GST-tag on APC16. Complexes were enriched by FLAG-affinity for BUBR1 or a donor UB mimic in some crosslinked complexes, with detailed purification procedures described in Supplemental Information.

Supplementary Material

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HIGHLIGHTS.

  • Cryo EM shows reciprocal and conformational regulation of APC/CCDC20 and MCC

  • MCC blocks APC/CCDC20 binding sites for substrates such as Cyclins and Securin

  • “Closed” APC/CCDC20-MCC blocks E2 UBE2C, as during the spindle checkpoint

  • “Open” APC/CCDC20-MCC directs ubiquitination of MCC as during checkpoint silencing

Acknowledgments

We thank M Brunner, KP Wu, JR Michael, DW Miller and S. Frase for advice and assistance.

Funding: Jane Coffin Childs Foundation, Leukemia & Lymphoma Society (NGB); Austrian Science Fund FWF–Hertha Firnberg Program (RQ); Japan Society for the Promotion of Science (MY); CIHR MOP#111149 and 136956 (SSS); Boehringer Ingelheim, the Austrian Science Fund (SFB-F34 and Wittgenstein award), the Austrian Research Promotion Agency (Headquarter grants FFG-832936 and FFG-852936, Laura Bassi Centre for Optimized Structural Studies grant FFG-840283), and the European Community (FP7/2007-2013, grant 241548, MitoSys) (JMP); DFG Sonderforschungsbereich 860 (HS); and ALSAC, NIH R37GM065930 and P30CA021765, and HHMI (BAS).

Footnotes

ACCESSION CODES

EMD-4021, EMD-4022, EMD-4023, EMD-4024, EMD-4025, EMD-4026, EMD-4027, EMD-4028, 5KHR.PDB, 5KHU.PDB

AUTHOR CONTRIBUTIONS

MY, RV, FW, SSS, JMP, HS and BAS planned and supervised the project. MY, RV, FW and RQ designed recombinant APC/C and MCC used herein. MY, RV, NGB, and WZ performed biochemical analyses. MY, RV and NGB made EM samples. PD and HS collected EM data, analyzed by MY, RV, BAS and HS with help from DH. MY, RV, JMP, HS and BAS prepared the paper.

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