MAD contortions: conformational dimerization boosts spindle checkpoint signaling

MAD contortions: conformational dimerization boosts spindle checkpoint signaling Marina Mapelli1,2 and Andrea Musacchio1,2 Almost two decades after their identification, the components of the mitotic checkpoint are finally revealing their structural secrets. The activation of Mad2, a central piece of the checkpoint protein machinery, is linked to the rare ability of this protein to adopt two distinct topologies. Current models of checkpoint function propose that the topological transition between the two states of Mad2 is rate limiting for checkpoint activation and is accelerated through a self-activation process based on the direct interaction of the two Mad2 conformers. These models add a molecular framework to an old theory that depicts kinetochores as catalysts in the generation of the mitotic checkpoint signal. Addresses 1 Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, I-20139 Milan, Italy 2 Research Unit of the Italian Institute of Technology (IIT) Foundation at the IFOM-IEO Campus, Via Adamello 16, I-20139 Milan, Italy Corresponding author: Musacchio, Andrea ([email protected])

Current Opinion in Structural Biology 2007, 17:716–725 This review comes from a themed issue on Proteins Edited by Edward Baker and Guy Dodson Available online 24th October 2007 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.08.011

which adds poly ubiquitin tags onto Securin and Cyclin B and targets them for proteasome-mediated proteolysis (Figure 1b). Before metaphase, when the sister chromatids are still searching for conditions of robust attachment to the mitotic spindle, the SAC restricts APC/C activity by negatively regulating its activator Cdc20 [4–6] (Figure 1a). There has been considerable progress towards the elucidation of the SAC mechanism [7,8]. At the heart of the SAC is Mad2 (mitotic arrest deficient 2), a conserved 200-residue protein. Mad2 has three unusual features, all of which have been framed by structural analysis [9–11,12,13,14]. First, Mad2 adopts two distinct topologies – open Mad2 (O-Mad2) and closed-Mad2 (C-Mad2) – differing in the position of secondary structure elements in the N- and C-terminal regions (Figure 2a and b; for a discussion on nomenclature of the Mad2 conformers, see Box 1). Second, C-Mad2 forms structurally equivalent complexes with two of its best-characterized ligands, the SAC protein Mad1 and the SAC target Cdc20, in which Mad1 and Cdc20 are entrapped by the so-called ‘safety belt’ or ‘seatbelt’ (Figure 2d). Binding of Mad1 and Cdc20 by C-Mad2 is intimately connected to the conversion from O-Mad2. Third, and perhaps more surprising, the O-Mad2 and C-Mad2 conformers engage in an asymmetric ‘conformational’ dimer (Figure 3). All evidence suggests that conformational dimerization with C-Mad2 stimulates the topological conversion of O-Mad2 required to bind Cdc20.

Mad2 conformers Introduction The switch-like transitions that mark the eukaryotic cell division cycle reflect the dialectics between a robust biochemical oscillator known as the cell cycle control system and feedback control mechanisms known as checkpoints [1,2]. The subject of this review – the control of the metaphase-to-anaphase transition by the spindle assembly checkpoint (SAC) – illustrates this principle (Figure 1). After all chromosomes have aligned at the spindle’s equator, the rapid activation of the protease Separase following destruction of its inhibitor Securin dissipates sister chromatid cohesion, in turn allowing sister chromatids to segregated to opposite spindle poles. Concomitantly, the destruction of Cyclin B removes the activity of the master mitotic kinase CDK1, driving cells out of mitosis. Both events require the activation of a multi-subunit Ubiquitin (Ub) ligase known as the anaphase promoting complex or cyclosome (APC/C) [3], Current Opinion in Structural Biology 2007, 17:716–725

Mad2 is a single domain protein belonging to the HORMA family [15]. The core of the HORMA fold contains three a-helices (aA, aB and aC) sandwiched between a 6-stranded b-sheet and an irregular b-hairpin [10] (Figure 2). In O-Mad2, the N-terminal b1-strand forms a parallel association to b5, while at the opposite edge of the sheet the C-terminal region folds as a b-hairpin. When Mad1 or Cdc20 are bound to Mad2, the chameleon b1-strand is displaced and assumes a helical conformation [9,13]. The Mad1 or Cdc20 ligands are incorporated into the body of Mad2, where they substitute the b7-b8 hairpin by augmenting the b-sheet at the b6 edge in an anti-parallel fashion (Figures 2 and 3). Concomitantly, the C-terminal hairpin moves across the b-sheet to replace the b1-strand, looping around the ligand with the b80 -b800 ‘safety belt’. Thus, the structurally invariant scaffold of the Mad2 fold consists of the three a-helices and the b4-b5-b6 strands. www.sciencedirect.com

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Figure 1

The metaphase-to-anaphase transition. (a) In prometaphase, the sister chromatids are in the process of attaching to spindle microtubules. Kinetochores that are devoid of microtubules recruit the Mad1:C-Mad2 complex. Mad1:C-Mad2 is the kinetochore receptor for cytosolic O-Mad2. A conformational dimer between C-Mad2 and O-Mad2 is formed, and the O-Mad2 molecule is ‘primed’ to bind Cdc20, changing its conformation into C-Mad2. The C-Mad2:Cdc20 complex binds BubR1:Bub3 to form the MCC, which binds to the APC/C and inactivates it. Note that Mad1 and Cdc20 have similar Mad2-binding motifs, but that in this model the two proteins do not compete because Mad1 always retains its associated C-Mad2, so that the Mad2 molecule eventually reaching Cdc20 is distinct from that bound to Mad1. (b) Anaphase (sister chromatid separation) is triggered when the MCC is disassembled from the APC/C, leading to the polyubiquitylation of Cyclin B and Securin and to their

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Figure 2

Structure and topology of Mad2 and p31comet. (a) Ribbon diagram of O-Mad2. The safety belt or seatbelt is blue, and the N-terminal region is red. (b) Ribbon diagram of Mad1-bound C-Mad2. The blue and red regions have changed their position, while the gray part of the structure is essentially invariant. When a ligand (in this case Mad1, colored gold) is entrapped under the safety belt, the C-Mad2 conformation is stabilized. Note that only a short segment of Mad1 is shown, but the protein has approximately 500 residues on the N-terminal side, and approximately 180 residues on the C-terminal side of the Mad2-binding motif (see also panel (d)). The entire structure of the Mad1:Mad2 core complex [9] is shown in Figure 3a. (c) Ribbon diagram of p31comet. The structure was determined in a complex with C-Mad2 [14] (not shown). Labeling of secondary structure elements in the p31comet core (grey) illustrates the similarity with Mad2. The safety belt of p31comet closes on the C-terminal segment (yellow), which occupies the pocket that in Mad2 is occupied by Mad1 or Cdc20. Despite the C-Mad2-like topology, however, p31comet acts as a structural mimic of O-Mad2 and binds C-Mad2 tightly [14]. (d) A surface view of the C-Mad2:Mad1 complex from the same view as in panel (b). The surface view illustrates the safety belt binding mechanism.

Mad1:C-Mad2 is the kinetochore receptor of O-Mad2 In late prophase–prometaphase, the SAC proteins are recruited to kinetochores, proteinaceous structures responsible for microtubule capture [5,16,17]. Recent analyses have identified the Kln1-Mis12-Ndc80 (KMN) network as a kinetochore receptor for spindle microtubules [18–20]. At unoccupied kinetochores, the KMN

network also contributes to recruit a tight Mad1:C-Mad2 complex [21,22], in the proximity of which Mad2 is thought to bind Cdc20. As Mad1 and Cdc20 bind the same Mad2 pocket and are therefore predicted to be competitive Mad2 ligands, Cdc20 may bind to Mad2 molecules dissociating from Mad1. A competition model, however, is in contrast to the notion that Mad1 is necessary for loading Mad2 onto Cdc20 in living cells [23]. The

(Figure 1 Legend Continued ) destruction by the proteasome. This triggers the activation of Separase, which causes loss of sister chromatid cohesion, and loss of CDK1 activity, which results in mitotic exit.

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Box 1 Nomenclature of Mad2 conformers and models of Mad2 conversion To harmonize the description of Mad2 conformers, Mapelli and collaborators and Yang and collaborators [12,14] agreed to adopt a common nomenclature. The name open-Mad2 (abbreviated as O-Mad2) will be used to describe structures previously described as O-Mad2 or N1-Mad2. The name closed-Mad2 (abbreviated as C-Mad2) will be used to describe structures previously referred to as C-Mad2, N2-Mad2 and N2’-Mad2 [8,9,10,11,12,13,14]. N2-Mad2 described an ‘empty’ C-Mad2 conformer devoid of Mad2 ligands [8,11]. The ‘empty’ form of C-Mad2 will be referred to as ‘unliganded C-Mad2’. Finally, the I-Mad2 notation was introduced to refer to the structural intermediate between the O-Mad2 and C-Mad2 states [12,14]. Three models of Mad2 activation in the spindle checkpoint have been discussed. The exchange model [11,33] proposes that Mad2 dissociates from Mad1 before it reaches Cdc20. As the evidence against this model is very strong, we do not discuss it further [7,8,25]. Two additional models of Mad2 activation have been proposed, the ‘Mad2 template’ and the ‘two-state’ models [7,8,25]. The elements of similarity in these models predominate over their differences. In both cases, a stable Mad1:C-Mad2 receptor recruits O-Mad2 to the kinetochore and acts as a platform for the Mad2 conversion required to bind Cdc20. The ‘two state’ model identifies in unliganded C-Mad2 the ‘active’ Mad2 species generated from conformational dimerization [8]. This aspect has not been discussed in detail in the ‘template model [25]. On the other hand, the ‘template model’ proposes that Cdc20:C-Mad2 might propagate the mechanism of conformational dimerization away from the Mad1:C-Mad2:-containing kinetochores, that is Cdc20:C-Mad2 might be endowed with the same catalytic properties of the Mad1: C-Mad2 complex. This aspect of the template model, which is based on the structural similarity of the Cdc20:C-Mad2 and Mad1:C-Mad2 complexes, has not been tested experimentally yet.

solution to this conundrum is that Mad2 is so tightly bound to Mad1 that it never dissociates from it within the timescale of mitotic checkpoint activation (1/2 h in human cells). As the cellular concentration of Mad1 in the cell is only 1/10–1/4 of the concentration of Mad2 (estimated to be around 100 nM, see ref [24] and discussion therein), there is plenty of free O-Mad2 available to bind Cdc20. The role of the constitutive C-Mad2 moiety of Mad1:C-Mad2 is to recruit O-Mad2 from the mitotic cytosol by means of conformational dimerization [25,26]. O-Mad2 then binds Cdc20 and turns into C-Mad2 (Figure 1a). Thus, the Mad1:C-Mad2 complex and Cdc20:C-Mad2 complex are structurally similar. Mad1:C-Mad2 is constitutive, while Cdc20:C-Mad2 is regulated: it forms early in mitosis thanks to the interaction of O-Mad2 with C-Mad2:Mad1, and it dissociates before anaphase to allow APC/C activation [24,26, 27,28,29,30].

Energetic and kinetic implications Both recombinant and endogenous Mad2 are predominantly folded as O-Mad2 [11,12], suggesting that this is the energetically more stable fold of Mad2 (Figure 4a). A puzzling observation is that purified recombinant O-Mad2 undergoes a partial and temperature-dependent www.sciencedirect.com

spontaneous conversion to C-Mad2 in vitro, even in the absence of Mad1 or Cdc20 ligands [11] (Figure 4a). While it is possible that the empty Mad2 (i.e. devoid of ligand and thus defined as unliganded C-Mad2) forms spontaneously from O-Mad2 because it is more stable, it seems more plausible that unliganded C-Mad2 is a less stable form of Mad2 whose generation is compensated by conformational dimerization with O-Mad2 (Figure 4a). Indeed, dimers containing O-Mad2 and unliganded C-Mad2 form rapidly in vitro from pure solutions of O-Mad2 unless special care is exercised to stabilize OMad2 during sample preparation to prevent its conversion into unliganded C-Mad2 [10,11,12,25,26,31,32]. If dimerization with O-Mad2 is important to form unliganded C-Mad2, then the impairment of the OMad2:C-Mad2 interaction should prevent unliganded C-Mad2 accumulation, a hypothesis that needs to be tested with mutants of appropriate penetrance (Table 1). The binding affinity of O-Mad2 for the Mad2-binding motifs of Mad1 or Cdc20 is 100 nM, and the affinity may be further increased by different mechanisms in living cells [4–6]. The mobile C-terminal and N-terminal regions of Mad2 are crucial determinants of the relative stability of the Mad2 conformers and also impinge on the ability of Mad2 to interact with Mad1 and Cdc20. For instance, removal of 10 C-terminal residues (Mad2DC10) locks Mad2 as O-Mad2 and renders it incompetent towards Cdc20 binding [10,33,34] (Table 1). A ‘locked’ O-Mad2 can also be obtained if the displacement of the b1-strand – a prerequisite for closure of the safety belt – is prevented [12]. Conversely, the deletion of the b1strand, or the substitution of a critical hydrophobic residue (Leu13) with other residues (Ala, Gln) in b1 stabilizes the unliganded C-Mad2 conformer [10,12,14,35] (Table 1), suggesting that removing b1 implies a large kinetic barrier and is rate limiting for the conversion of OMad2 to C-Mad2. In vitro, the slow basal rate of binding of Mad2 to Cdc20 is made significantly slower if conformational dimerization is impaired (7–50 times for different mutants; reference [11], and unpublished results by M. Simonetta, R. Manzoni, M.M, A.M., and A. Ciliberto). As conformational dimerization is also essential to load Mad2 onto Cdc20 in living cells [25,26], it can be asked if conformational dimerization of Mad2 has a ‘catalytic’ role on the Mad2 conversion [36,37].

Structural bases of conformational dimerization In support of a catalytic role for conformational dimerization of Mad2, NMR chemical shift perturbation experiments revealed that O-Mad2 undergoes a global conformational change upon binding to the rigid scaffold of C-Mad2 [35]. Thus, after docking on Mad1-bound C-Mad2, O-Mad2 may turn into an intermediate (I-Mad2) with enhanced Cdc20-binding capabilities relative to O-Mad2 (Figure 4b). The crystallographic Current Opinion in Structural Biology 2007, 17:716–725

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Figure 3

Conformational dimerization of Mad2. (a) The Mad1:C-Mad2 core complex [9] is a 2:2 tetramer. Mad1 is colored gold, while C-Mad2 is grey, red and blue like in Figure 2. (b) In principle, the Mad1:Mad2 core complex has two identical binding sites for O-Mad2. The model shown in this panel depicts the Mad1:C-Mad2 2:2 complex bound to two molecules of O-Mad2. The model is based on the crystal structures of the Mad1: C-Mad2 core complex [9] and of the O-Mad2:C-Mad2 conformational dimer [12] shown in panels (a) and (c). (c) and (d) Two orthogonal views of the O-Mad2:C-Mad2 conformational dimer [12]. The asymmetry of the complex can be easily grasped from the position of the safety belt (blue) in the O-Mad2 and C-Mad2 moieties, and from the reciprocal orientation of equivalent helices in the two protomers.

structure of the Mad2 conformational dimer [12] (Figure 3c and d), does not reveal what I-Mad2 looks like, as the conformation of O-Mad2 in this structure was artificially stabilized by the Mad2LL mutation (Table 1). Thus, the structure likely represents a snapshot of the initial docking of O-Mad2 onto C-Mad2. In the Mad2 conformational dimer [12], the aC-helices of both protomers and the C-terminal hairpin of C-Mad2 predominate at the dimer interface. The involvement of the b80 -b800 -hairpin of C-Mad2, one of the mobile elements of the Mad2-fold, is a major element of asymmetry at the interface (Figure 3c and d). The aC-helices of both protomers are also engaged at the interface, but they face different environments and coordinate different interaction networks on the two sides. This organization identifies residues in the aC-helix (such as Phe141 of human Mad2) that contribute to the binding interface of Current Opinion in Structural Biology 2007, 17:716–725

only one protomer (C-Mad2) but not the other (O-Mad2) (Table 1). These ‘conformationally selective’ mutants may prove useful to knock out the dimerization features of Mad2 on distinct protomers [12]. In summary, the asymmetry of the O-Mad2:C-Mad2 interface is probably required to adapt O-Mad2 onto the rigid C-Mad2 scaffold and promote its conformational change [35] into a form that is better suited to bind Cdc20 (I-Mad2 in Figure 4b). I-Mad2 might have ‘floppy’ N- or C-terminal tails ready to be restructured if Cdc20 were rapidly encountered. The ‘two-state’ model of Mad2 activation proposes a possible twist to this model (Box 1). In particular, I-Mad2 may first turn into unliganded C-Mad2 (see above), possibly in the form of a symmetric dimer (refs. [8,11] and H. Yu and X. Luo, personal communication). Then, the unliganded C-Mad2 www.sciencedirect.com

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Figure 4

Energetic and kinetic implications of the Mad2 conversion. (a) In the absence of Mad1 and Cdc20, pure preparations of wild type O-Mad2 convert into equal mixtures of unliganded C-Mad2 and O-Mad2 that engage in a dimer (see text for additional details). If unliganded C-Mad2 is a high-energy state relative to O-Mad2, its formation may be compensated by conformational dimerization with the remaining O-Mad2. It is possible that the reaction shown in this panel is self-catalyzed by unliganded C-Mad2, but we do not discuss this possibility in detail here (see ref [32]). We postulate that the ‘transition state’ I-Mad2 is a protein in which certain secondary structure elements, and in particular the b1-strand, have been displaced from the position they occupy in O-Mad2 to favor the structural conversion to C-Mad2. (b) A simple model for how O-Mad2 may bind Cdc20 with the help of Mad1-bound C-Mad2. The intermediate I-Mad2 is formed from O-Mad2 bound to the Mad1: C-Mad2 core complex. As several bonds must be broken to displace the b1-strand, the activation energy is high. The transition state I-Mad2 binds Cdc20 and the Cdc20:C-Mad2 complex is formed. In this model, the Mad1:C-Mad2 complex acts as a catalyst by reducing the activation energy required to create I-Mad2. (c) A more complex model, similar to that proposed in the ‘two state model’ (ref. [8] and Box 1). In this case, passage through the transition state I-Mad2 leads to the formation of unliganded C-Mad2, as in panel (a). As in the example of panel (b), this part of the reaction is catalyzed by the Mad1:C-Mad2 complex. Later, unliganded C-Mad2 binds Cdc20. For this, it will have to re-open the safety belt by loosening the interaction of the b80 -b800 hairpin with the Mad2 hydrophobic core, creating the transition state I’-Mad2. For unknown reasons, the dimer between unliganded C-Mad2 and O-Mad2 shown in panel (a) does not appear to form in living cells. All schemes in panels (a–c) are hypothetical but can provide a useful rationalization to the Mad2 conversion problem.

will bind to Cdc20. In vitro, unliganded C-Mad2 is a more potent APC/C inhibitor than O-Mad2 [11]. As the safety belt is closed in unliganded C-Mad2, the conditions for unliganded C-Mad2 to be the active Mad2 species are 1) that the b80 –b800 hairpin, which replaces the b1-strand of O-Mad2, ‘breaths’ (I’-Mad2 in Figure 4c) to allow Cdc20 to fit snugly under the belt, and 2) that the time scale of such breathing is faster relative to that characterizing the displacement of b1. Accurate kinetic measurements in finely reconstituted biochemical systems will be instrumental to clarify this point.

Targeting Cdc20 to inhibit the APC C-Mad2:Cdc20 associates with the additional SAC proteins BubR1 (known as Mad3 in fungi) and Bub3 to form the Mitotic Checkpoint Complex (MCC) that is currently regarded as the actual molecular species inhibiting the APC/C [38,39]. Recent observations suggest that rather than sequestering Cdc20, the MCC assembles on the APC/C, possibly via a pseudo-substrate mechanism mediated by BubR1/Mad3 [29,30,40,41] (Figure 1). The structural bases of these critical interactions are currently unknown. Recent electron microscopy reconstructions of vertebrate and yeast APC/C have begun to elucidate the molecular organization of this multi-subunit enzyme [42,43] and may, in the future, www.sciencedirect.com

help to reveal the site of binding of the MCC onto the APC/C.

p31comet: a new embellishment of the HORMA domain It follows from the idea that the uncatalyzed interaction of Mad2 with Cdc20 has slow forward and backward kinetics that mechanisms actively dissociating the Mad2:Cdc20 complex might exist to insure rapid progression into anaphase once all chromosomes have attached to the spindle. The p31comet protein (initially named CMT2) was first identified as a binding partner of Mad2 regulating mitotic exit [44]. Subsequently, p31comet was shown to associate selectively with C-Mad2 and to compete with its ability to form conformational dimers with O-Mad2 [24,35,45]. Based on these biochemical properties, p31comet is believed to induce anaphase by blocking the dimerization-dependent association of Mad2 with Cdc20 when the SAC is satisfied. The structure of the p31comet:C-Mad2 complex reveals that p31comet is a structural mimic of Mad2 that folds as a HORMA domain [14]. The structure of the p31comet: C-Mad2 complex closely resembles that of the Mad2 conformational dimer [12,14]. A striking difference, however, is that the topology of p31comet is similar to that Current Opinion in Structural Biology 2007, 17:716–725

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Table 1 Properties of Mad2 mutants Mad2 species

Description

Preferred conformer a

Conformational dimerization b

Binding to Cdc20 or Mad1 b

SAC proficiency

References

PDB entry

O-Mad2 Locked as O-Mad2 C-Mad2 C-Mad2

Viable Viable Viable

Yes No Yes Yes

Compromised TBD TBD

[11,12,31] [10,25,31] [35] [12]

1KLQ, 2V64 1DUJ

Locked as O-Mad2 C-Mad2 C-Mad2 O-Mad2 C-Mad2 No evident preference Locked as O-Mad2

Viable Viable Viable Symmetrically affected Affected on C-Mad2 Symmetrically affected Affected

No Yes Yes Yes Yes Yes No

TBD TBD TBD Compromised Compromised Compromised Compromised

[12] [12,14] [12,14] [9,25,26,11] [25,26] [25] [25]

2V64 2YQF

Mad2 T140A Mad2 F141A Mad2 R184A

None Residues 196–205 deleted Residues 1–15 deleted Residues 1–15 and 109–117 deleted Residues 109–117 deleted Point mutation Point mutation Point mutation Point mutation Double point mutation Double point mutation and residues 196–205 deleted Point mutation Point mutation Point mutation

O-Mad2 O-Mad2 O-Mad2

Symmetrically affected Affected on C-Mad2 Affected on C-Mad2

Yes Yes Yes

[35] [35] [35]

Mad2 W100Y Mad2 V193N

Point mutation Point mutation

O-Mad2 Locked as O-Mad2

Viable Viable

Yes No

Compromised Compromised Partly compromised TBD TBD

Mad2 wt Mad2DC Mad2DN15 Mad2DN15-LL Mad2 LL Mad2 L13A Mad2 L13Q Mad2 R133A Mad2 Q134A Mad2R133E-Q134A Mad2DC-R133E-Q134A

1GO4, 1S2H

[12] [12]

TBD: To be determined. The favorite conformation adopted by each Mad2 construct (expressed at 168C and purified at 48C) can be assigned based on fractionation from an anion-exchange column [11,12]. b The ability of Mad2 and Mad2 mutants to form dimers and to bind Cdc20 can be assessed in a pull-down assay, in which a GST fusion of the Mad2-binding domain of Cdc20 immobilized on solid phase is used as a bait to test the ability of Mad2 mutants to bind Cdc20 and form C-Mad2. The C-Mad2 moiety is then tested for its ability to bind O-Mad2 (see ref. [35] for details). The ability of the Mad2 mutants to form conformational dimers can be further addressed by analytical size exclusion chromatography (again, see ref. [35] for details). a

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of C-Mad2 (Figure 2c): the C-terminal segment of p31comet inserts as a pseudo-substrate in a pocket that is equivalent to the one where Cdc20 and Mad1 bind Mad2 [14]. Whether the same pocket can also accept binding motifs from other proteins is currently unclear, and also unclear is whether p31comet adopts topologies other than that revealed by its crystal structure with C-Mad2. The same question should be extended to additional HORMA family members, such as Rev7 and Hop1 [15]. The surface-exposed residues mediating the conformational dimerization of Mad2 are divergent in Rev7 and Hop1, suggesting that at least this property of Mad2 is absent in Rev7 and Hop1. As no homologue of p31comet has been identified in fungi, the function of p31comet might have evolved to control the greater complexity of the SAC network in higher eukaryotes [6]. Besides contrasting the O-Mad2:C-Mad2 association, p31comet has been proposed to act synergistically with UbcH10 to promote the active dissociation of C-Mad2 from Cdc20 before anaphase [46]. USP44, a deubiquitinating enzyme, has been implicated as a possible negative regulator of this pathway [28].

undergo an equally dramatic change of topology in association with their cleavage [50]. What is new about Mad2 is that the topological conversion of O-Mad2 seems to be catalyzed by C-Mad2, the product of the conversion. We hope that a structural characterization of the I-Mad2 intermediate will shed light on the protein folding implications of this beautiful two-state mechanism. Even more challenging will be the design of experiments addressing the Mad2 mechanism in living cells and in appropriately reconstituted systems.

Acknowledgements Research in A. Musacchio’s laboratory is funded by the Association for International Cancer Research (AICR), the Telethon Foundation, the EU FP6 program contracts 3D-Repertoire and Mitocheck, the Italian Association for Cancer Research (AIRC), the Fondo di Investimento per la Ricerca di Base (FIRB), the Italian Ministry of Health, and the Fondazione Cariplo. Because of space restraints, we only quoted a small number of contributions. We apologize to all authors whose work was not cited. We are very grateful to Hongtao Yu, Xuelian Luo, Andrea Ciliberto, Marco Simonetta, Romilde Manzoni and to the members of the Musacchio laboratory for many helpful discussions, and to Hongtao Yu and Xuelian Luo for communicating results before publication.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

Conclusions The asymmetric dimerization of O-Mad2 with Mad1bound C-Mad2 at unoccupied kinetochores is a very striking feature of the SAC network. Although the precise mechanism whereby this interaction favors the accumulation of Mad2:Cdc20 requires further investigations, that its significance is to facilitate the conversion of cytosolic O-Mad2 into C-Mad2 bound to Cdc20 seems to be rooted in the biological data [6]. This model identified Mad1-bound C-Mad2 as a template for O-Mad2 conversion into a Cdc20-bound C-Mad2 copy [25]. At least in principle, Cdc20:C-Mad2 complexes could also assemble conformational dimers with O-Mad2, providing positive feedback to the propagation of the checkpoint signal away from kinetochores [47]. There is at present no direct proof that this additional amplification strategy exists. Cases of asymmetric dimers of identical protein chains are well documented. For instance, structurally equivalent kinase domains of the EGF receptor dimerize via an asymmetrical C-lobe to N-lobe interaction that is critical for the activation of kinase activity in one of the subunits [48]. The protease caspase 9 assembles in dimers of slightly different conformers of which only one is catalytically competent [49]. The conformational dimerization of Mad2 appears to be a transient interaction between an O-Mad2 subunit and a spatially localized rigid scaffold—the Mad1:C-Mad2 complex. The interaction is required to trigger the conformational change associated with the binding of O-Mad2 to its target Cdc20. The novelty here is not that Mad2 adopts two distinct topologies, as the serpins have been shown to www.sciencedirect.com

 of special interest  of outstanding interest 1.

Hartwell LH, Weinert TA: Checkpoints: controls that ensure the order of cell cycle events. Science 1989, 246:629-634.

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Morgan DO: Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 1997, 13:261-291.

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Peters JM: The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 2006, 7:644-656.

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Yu H: Cdc20: A WD40 Activator for a cell cycle degradation machine. Mol Cell 2007, 27:3-16.

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Taylor SS, Scott MI, Holland AJ: The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation. Chromosome Res 2004, 12:599-616.

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Musacchio A, Salmon ED: The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 2007, 8:379-393.

7. Nasmyth K: How do so few control so many? Cell 2005,  120:739-746. This review article presents a very comprehensive discussion of the Mad2 conformational changes and of its biological implications. 8. 

Yu H: Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model. J Cell Biol 2006, 173:153-157. The author presents the Mad2 ‘two state’ model and discusses its differences and similarities to the ‘template’ model. 9.

Sironi L, Mapelli M, Knapp S, Antoni AD, Jeang K-T, Musacchio A: Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint. EMBO J 2002, 21:2496-2506.

10. Luo X, Fang G, Coldiron M, Lin Y, Yu H, Kirschner MW, Wagner G: Structure of the mad2 spindle assembly checkpoint protein and its interaction with cdc20. Nat Struct Biol 2000, 7:224-229. 11. Luo X, Tang Z, Xia G, Wassmann K, Matsumoto T, Rizo J, Yu H:  The Mad2 spindle checkpoint protein has two distinct natively folded states. Nat Struct Mol Biol 2004, 11:338-345. Current Opinion in Structural Biology 2007, 17:716–725

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In this paper, the authors propose that unliganded C-Mad2 is the active Mad2 species that binds Cdc20. They show that unliganded C-Mad2 is a stronger APC/C Ub-ligase inhibitor compared to O-Mad2. 12. Mapelli M, Massimiliano L, Santaguida S, Musacchio A: The Mad2  conformational dimer: structure and implication for the spindle assembly checkpoint. Cell 2007, in press. Together with the structure described in Ref. 14, the crystal structure of the O-Mad2:C-Mad2 dimer described in this paper provides the bases to understand conformational dimerization at the molecular level. 13. Luo X, Tang Z, Rizo J, Yu H: The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Molecular Cell 2002, 9:59-71. 14. Yang M, Li B, Tomchick DR, Machius M, Rizo J, Yu H, Luo X:  p31comet blocks Mad2 activation through structural mimicry. Cell 2007, in press. The authors describe the structure of the complex of p31comet with C-Mad2, and report that p31comet folds as a HORMA domain. Together with Ref. 12, this paper describes the structural bases for conformational dimerization of Mad2 15. Aravind L, Koonin EV: The HORMA domain: a common structural denominator in mitotic checkpoints, chromosome synapsis and DNA repair. Trends Biochem Sci 1998, 23:284-286. 16. Ciferri C, Musacchio A, Petrovic A: The Ndc80 complex: Hub of kinetochore activity. FEBS Lett 2007, 581:2862-2869. 17. Cleveland DW, Mao Y, Sullivan KF: Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 2003, 112:407-421. 18. Cheeseman IM, Chappie JS, Wilson-Kubalek EM, Desai A: The conserved KMN network constitutes the core microtubulebinding site of the kinetochore. Cell 2006, 127:983-997. 19. DeLuca JG, Gall WE, Ciferri C, Cimini D, Musacchio A, Salmon ED: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 2006, 127:969-982. 20. Wei RR, Al-Bassam J, Harrison SC: The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment. Nat Struct Mol Biol 2007, 14:54-59. 21. Martin-Lluesma S, Stucke VM, Nigg EA: Role of hec1 in spindle checkpoint signaling and kinetochore recruitment of mad1/ mad2. Science 2002, 297:2267-2270. 22. DeLuca JG, Howell BJ, Canman JC, Hickey JM, Fang G, Salmon ED: Nuf2 and hec1 are required for retention of the checkpoint proteins mad1 and mad2 to kinetochores. Curr Biol 2003, 13:2103-2109. 23. Hwang LH, Lau LF, Smith DL, Mistrot CA, Hardwick KG, Hwang ES, Amon A, Murray AW: Budding yeast Cdc20: a target of the spindle checkpoint. Science 1998, 279:1041-1044. 24. Vink M, Simonetta M, Transidico P, Ferrari K, Mapelli M, De  Antoni A, Massimiliano L, Ciliberto A, Faretta M, Salmon ED et al.: In Vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics. Curr Biol 2006, 16:755-766. The authors describe the biochemical reconstitution of the mechanism of kinetochore recruitment of O-Mad2, and show that the reconstituted system has kinetic properties that are very similar to those of the endogenous cellular reaction. 25. De Antoni A, Pearson CG, Cimini D, Canman JC, Sala V, Nezi L,  Mapelli M, Sironi L, Faretta M, Salmon ED et al.: The mad1/mad2 complex as a template for mad2 activation in the spindle assembly checkpoint. Curr Biol 2005, 15:214-225. Based on the identification of the Mad1:C-Mad2 complex as the kinetochore receptor of O-Mad2, the authors present the ‘template’ model. 26. Nezi L, Rancati G, De Antoni A, Pasqualato S, Piatti S,  Musacchio A: Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae. J Cell Biol 2006, 174:39-51. Demonstration that Mad2 mutants impaired in conformational dimerization fail to reconstitute the checkpoint in Saccharomyces cerevisiae’s mad2 deletion strains. Current Opinion in Structural Biology 2007, 17:716–725

27. Shah JV, Botvinick E, Bonday Z, Furnari F, Berns M,  Cleveland DW: Dynamics of centromere and kinetochore proteins; implications for checkpoint signaling and silencing. Curr Biol 2004, 14:942-952. A fluorescence recovery after photobleaching (FRAP) analysis identifies a non-exchanging Mad2 fraction. Later, reference 24 identified the nonexchanging component as Mad1-bound C-Mad2. 28. Stegmeier F, Rape M, Draviam VM, Nalepa G, Sowa ME, Ang XL,  McDonald ER, 3rd, Li MZ, Hannon GJ, Sorger PK et al.: Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 2007, 446:876-881. The authors describe the involvement of the deubiquitinating enzyme USP44 in a network that positively regulates the spindle assembly checkpoint. The paper should be considered together with ref [46]. 29. Braunstein I, Miniowitz S, Moshe Y, Hershko A: Inhibitory factors  associated with anaphase-promoting complex/cylosome in mitotic checkpoint. Proc Natl Acad Sci U S A 2007, 104:4870-4875. The authors demonstrate that the duration of the lag phase required for APC/C activation in cell extracts correlates with the dissociation of the MCC from the APC/C. 30. Morrow CJ, Tighe A, Johnson VL, Scott MI, Ditchfield C, Taylor SS:  Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20. J Cell Sci 2005, 118:3639-3652. The authors try to correlate the formation of an MCC:APC/C complex with different stages and perturbations of mitosis and show that the association of the MCC with the APC/C varies during mitosis. 31. Fang G, Yu H, Kirschner MW: The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev 1998, 12:1871-1883. 32. DeAntoni A, Sala V, Musacchio A: Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2. Philos Trans R Soc Lond B Biol Sci 2005, 360:637-647, discussion 447–638. 33. Sironi L, Melixetian M, Faretta M, Prosperini E, Helin K, Musacchio A: Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint. EMBO J 2001, 20:6371-6382. 34. Chen RH, Brady DM, Smith D, Murray AW, Hardwick KG: The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins. Mol Biol Cell 1999, 10:2607-2618. 35. Mapelli M, Filipp FV, Rancati G, Massimiliano L, Nezi L, Stier G,  Hagan RS, Confalonieri S, Piatti S, Sattler M et al.: Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet). Embo J 2006, 25:1273-1284. By demonstrating that p31comet binds to the same C-Mad2 surface to which O-Mad2 also binds, the authors provide a molecular explanation to the observation that p31comet is a SAC inhibitor. 36. Shah JV, Cleveland DW: Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell 2000, 103:997-1000. 37. Gorbsky GJ, Kallio M, Daum JR, Topper LM: Protein dynamics at the kinetochore: cell cycle regulation of the metaphase to anaphase transition. FASEB J 1999, 13(Suppl. 2): S231-S234. 38. Hardwick KG, Johnston RC, Smith DL, Murray AW: MAD3 Encodes a Novel Component of the Spindle Checkpoint which Interacts with Bub3p, Cdc20p, and Mad2p. J Cell Biol 2000, 148:871-882. 39. Sudakin V, Chan GK, Yen TJ: Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J Cell Biol 2001, 154:925-936. 40. Burton JL, Solomon MJ: Mad3p, a pseudosubstrate inhibitor of  APCCdc20 in the spindle assembly checkpoint. Genes Dev 2007, 21:655-667. Together with reference 41, this paper suggests that that Mad3 KEN boxes might be responsible for the association of the MCC with the APC/C. 41. King EM, van der Sar SJ, Hardwick KG: Mad3 KEN boxes  mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint. PLoS ONE 2007, 2:e342. www.sciencedirect.com

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42. Dube P, Herzog F, Gieffers C, Sander B, Riedel D, Muller SA, Engel A, Peters JM, Stark H: Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C. Mol Cell 2005, 20:867-879. 43. Passmore LA, Booth CR, Venien-Bryan C, Ludtke SJ, Fioretto C, Johnson LN, Chiu W, Barford D: Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation. Mol Cell 2005, 20:855-866. 44. Habu T, Kim SH, Weinstein J, Matsumoto T: Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J 2002, 21:6419-6428. 45. Xia G, Luo X, Habu T, Rizo J, Matsumoto T, Yu H: Conformation specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint. EMBO J 2004, 23:3133-3143. The authors demonstrate that p31comet binds selectively to the C-Mad2 conformer. 46. Reddy SK, Rape M, Margansky WA, Kirschner MW:  Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 2007, 446:921-925.

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Together with reference [28], this paper shows that a dynamic balance of ubiquitination and deubiquitination contributes to the generation of the switch-like transition controlling anaphase entry. 47. Doncic A, Ben-Jacob E, Barkai N: Evaluating putative  mechanisms of the mitotic spindle checkpoint. Proc Natl Acad Sci U S A 2005. The first attempt to provide a mathematic description of the spindle assembly checkpoint. 48. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J: An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 2006, 125:1137-1149. 49. Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS: Dimer formation drives the activation of the cell death protease caspase 9. Proc Natl Acad Sci U S A 2001, 98:14250-14255. 50. Brown JH: Breaking symmetry in protein dimers: designs and functions. Protein Sci 2006, 15:1-13.

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