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To maintain genetic stability, cells first duplicate their chromosomesprecisely once per cell division and then partition the duplicated sisterchromatids evenly during mitosis. The sister chromatids are heldtogether by the cohesin protein complex. At the juncture of chromo-some segregation, the anaphase-promoting complex or cyclosome(APC/C) attaches polyubiquitin chains to the securin protein.Degradation of ubiquitinated securin relieves its inhibition of the pro-tease called separase. Active separase then cleaves the Scc1 subunit ofcohesin, allowing the spindle microtubules to pull the separated chro-matids to opposite poles of the cell (for a review, see ref. 1). Theprocess of chromosome segregation is monitored by a surveillancemechanism called the spindle checkpoint2. In response to a singlechromatid not properly attached to the mitotic spindle in a mitoticcell, the spindle checkpoint blocks the ubiquitin ligase activity ofAPC/CCdc20, stabilizes securin, prevents cleavage of cohesin, preservessister chromatid cohesion and delays separation of sister chro-matids1–3. To explain the fine sensitivity of this checkpoint, it has beensuggested that the kinetochore of the lone misaligned chromatid gen-erates a diffusible inhibitory signal that is distributed throughout thecell to inhibit APC/C not associated with this kinetochore2,4. Althoughthe nature of this diffusible ‘wait anaphase’ signal remains to be estab-lished, the mitotic checkpoint complex (MCC) containing BubR1(Mad3), Bub3, Mad2 and Cdc20, and its subcomplexes, are attractivecandidates for this signal2,3,5–9. In particular, binding between Mad2and Cdc20, possibly as part of MCC, is required for the proper func-tion of the spindle checkpoint and is greatly enhanced upon check-point activation3. Mad2 also associates with another checkpoint

protein, Mad1, throughout the cell cycle, and Mad1 is required for theestablishment of the Mad2-Cdc20 interaction in vivo10–12. In contrast,two recently discovered mechanisms––binding of Cmt2 to Mad2 andphosphorylation of the C terminus of Mad2––seem to negatively regu-late Mad2 (refs. 13,14).

Mad2 recognizes similar short peptide motifs in Mad1 (its upstreamregulator) and Cdc20 (its downstream target)11. We have previouslydetermined the structure of Mad2 in complex with a peptide ligand,MBP1, which was identified through phage display and mimics theMad2-binding motifs of both Mad1 and Cdc20 (ref. 11). The structureof Mad2 bound to a 100-residue fragment of Mad1 has also beenreported15. These structural studies demonstrate that binding of Mad1or Cdc20 triggers a similarly marked conformational change of Mad2(refs. 11,15,16; see below). The Mad2-binding motif of Mad1 or Cdc20is incorporated as a central strand into the main β-sheet of Mad2. TheC-terminal region of the free Mad2 rearranges into a β-hairpin (β8 and β9) and reaches across the protein to displace the N-terminalβ1 strand, which becomes an extra turn in helix A. Because the Mad2-binding motifs of Mad1 and Cdc20 are located in the central regions ofthese proteins, the translocation of the β8-β9 hairpin of Mad2 effec-tively traps Mad1 or Cdc20 in a manner analogous to the seat belts ofautomobiles11,15. For Mad1 or Cdc20 to dissociate from or reassociatewith Mad2, the β8-β9 hairpin needs to detach from the main β-sheet.This unusual binding mechanism may prolong the lifetimes of theMad2-containing protein complexes3,17.

Because Mad1 and Cdc20 bind to similar sites on Mad2, binding of Mad1 and Cdc20 to Mad2 is mutually exclusive11,18. Yet the

Departments of 1Pharmacology and 2Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.3Laboratoire de Biologie Cellulaire du Développement, UMR7622, CNRS, Université Pierre et Marie Curie, 9 Quai Saint Bernard, 75005 Paris, France. 4RadiationBiology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan. 5These authors contributed equally to this work. Correspondence should be addressedto J.R. (jose@arnie.swmed.edu) or H.Y. (hongtao.yu@utsouthwestern.edu).

Published online 14 March 2004; doi:10.1038/nsmb748

The Mad2 spindle checkpoint protein has two distinctnatively folded statesXuelian Luo1,2,5, Zhanyun Tang1,5, Guohong Xia1,5, Katja Wassmann3, Tomohiro Matsumoto4, Josep Rizo1,2 &Hongtao Yu1

The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuringthe fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target ofthis checkpoint, the ubiquitin protein ligase APC/CCdc20. We now show that without cofactor binding or covalent modificationMad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 hasbeen determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutantthat specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvertslowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggestthat the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.

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Mad1-Mad2 interaction is required for the binding of Mad2 to Cdc20upon checkpoint activation in vivo10–12. To resolve this paradox, we have previously suggested that Mad2 dissociated from theMad1–Mad2 complex might transiently retain an ‘activated’ confor-mation more compatible with binding to Cdc20 (refs. 3,11). Thishypothesis explains the Mad1-assisted formation of the Mad2–Cdc20complex in vivo. Here we demonstrate the existence of an activatedform of Mad2, report its three-dimensional structure, provide evi-dence to suggest that this form of Mad2 might be involved in check-point signaling in living cells, and show that the formation of thisactive form of Mad2 is facilitated by a fragment of Mad1 in vitro.

RESULTSMad2 has two distinct native foldsTo obtain the activated form of Mad2, we took advantage of the factthat bacterially expressed Mad2 protein exists in both monomericand dimeric forms19. The dimeric form ofMad2 can further aggregate to yield higher-order oligomers at high concentrations19.The dimeric Mad2 was more potent ininhibiting APC/C in Xenopus laevis eggextracts than was the monomeric form, sug-gesting that the conformation of the Mad2dimer might resemble that of the activatedMad2 (ref. 19). Recently, the R133A mutantof Mad2 (Arg133 is a conserved surfaceresidue on helix C) has been shown to beexclusively monomeric in vitro and yetretains full biological activity in vivo18, sug-gesting that dimerization of Mad2 intrinsi-cally might not be required for checkpointsignaling. We reasoned that the R133A muta-tion might have uncoupled the conforma-tional change and dimerization processes ofMad2. This allowed us to test the hypothesisthat a conformational change of Mad2 mightbe required for checkpoint activation.

Consistent with the earlier findings18, theMad2 R133A protein expressed in bacteriawas monomeric. However, it eluted from an

anion exchange column as two well-separated peaks (Fig. 1a). The twopeaks of Mad2 R133A were concentrated separately, and 1H-15NHSQC spectra were acquired on these samples (Fig. 1b). Based on thepatterns of the NMR spectra, the low-salt peak of Mad2 R133A had aconformation identical to the previously determined structure of thefree Mad2 monomer16 (Fig. 1b, black contours). To facilitate discus-sion, we refer to this conformation of Mad2 as N1, for native fold 1.The 1H-15N HSQC spectrum of the high-salt peak of Mad2 R133A(Fig. 1b, cyan contours) was highly similar to that of the ligand-boundform of Mad2 (see Supplementary Fig. 1 online)11, which we refer toas N2, for native fold 2. Based on mass spectrometry, both species ofMad2 had the same predicted mass for unmodified Mad2, thusexcluding the involvement of covalent modification. Notably, N1-Mad2 R133A spontaneously converted to the N2 form in the NMRtube at 30 °C overnight but did not do so at 4 °C, the temperature atwhich this protein was purified (see below). At equilibrium, the molar

Figure 1 Mad2 R133A has two natively folded monomeric conformers. (a) The UV trace (top panel) and Coomassie blue–stained SDS-PAGE analysis (bottompanel) of the Mono-Q column fractions of Mad2 R133A. Fractionation was done at 4 °C; the two forms of Mad2 had not yet reached equilibrium. The saltconcentrations of the two peaks are labeled. (b) Overlay of the 1H-15N HSQC spectra of N1-Mad2 R133A (black) and N2-Mad2 R133A (in cyan). (c) Overlayof the 1H-15N HSQC spectrum of N2-Mad2 R133A (cyan) and the 1H-15N TROSY-HSQC spectrum of the 2H-15N-labeled wild-type N2-Mad2 dimer (red).

Figure 2 Ribbon drawing of the structures of N1-Mad2 (left)16, N2-Mad2 R133A (middle, this study),and ligand-bound Mad2 (right)11. The β-strands are blue, α-helices green, and loops ivory. The non-natural Mad2-binding peptide, MBP1, is red. The structural elements of Mad2 that undergo majorchanges between the N1 and N2 conformers or upon peptide binding are yellow and orange. Thestrands are numbered 1–8 and the helices are labeled A–C in the N1-Mad2 structure. MBP1 is labeledas β1′ in the Mad2–MBP1 complex. The secondary structure elements of the N2-Mad2 R133A andMad2–MBP1 structures are labeled similarly to those of N1-Mad2 with the exception of β9, which isunstructured in N1-Mad2. Arg133 is shown as ball-and-stick in the N1-Mad2 structure. Generatedwith MolScript40 and Raster3D41.

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ratio of N2/N1 was ∼ 8:1 in a buffer containing 50 mM phosphate,pH 6.8, 300 mM KCl and 1 mM DTT at 30 °C, as determined from the intensities of the 1H-15N HSQC crosspeaks corresponding to N1-Mad2 and N2-Mad2, and by integration of the UV traces of the Q-column peaks of the two conformers (data not shown). The equi-librium constant was very similar in a buffer containing 50 mM Tris,pH 7.7, 150 mM KCl and 1 mM DTT at 30 °C, indicating that the equi-librium of the two Mad2 conformers is not substantially altered bychanges in pH and salt concentration. Finally, refolding of chemicallydenatured Mad2 R133A at 4 °C resulted in the formation of both N1and N2 conformers with a molar ratio of 1:2 (Supplementary Fig. 2online). Because N1-Mad2 R133A does not undergo the N1-N2 con-version at 4 °C with appreciable rates and yet ∼ 70% of the refoldedMad2 R133A adopts the N2 conformation at 4 °C, it is unlikely thatN1-Mad2 is an obligatory kinetic folding intermediate of N2-Mad2.Therefore, Mad2 R133A adopts two distinct monomeric conforma-tions at equilibrium under native conditions.

To rule out the possibility that the two-state behavior of Mad2R133A was caused by the R133A mutation, we also examined the wild-type Mad2 protein. The wild-type Mad2 monomer purified from bac-teria exhibited the N1 conformation as judged by its 1D and 1H-15NHSQC spectra16 (see below). Upon overnight incubation at 30 °C, itdimerized (see below). Furthermore, the 1H-15N TROSY-HSQC spec-trum of the wild-type Mad2 dimer (Fig. 1c, red contours) was similarto the 1H-15N HSQC spectrum of N2-Mad2 R133A (Fig. 1c, cyan con-tours), indicating that the wild-type Mad2 dimer adopts the N2 con-formation. Therefore, wild-type Mad2 also behaves as a two-stateprotein and can undergo the N1-N2 structural transition. However,wild-type N2-Mad2 is prone to dimerization. It is likely that helix C ispart of the dimer interface and that the R133A mutation disruptsdimerization of N2-Mad2, thus preserving the monomeric N2 form.The monomeric Mad2 R133A mutant can adopt both the N1 and N2conformations, also indicating that dimerization of Mad2 is notrequired for the N1-N2 conformational change.

We determined the structure of N2-Mad2 R133A using multidi-mensional NMR spectroscopy (Fig. 2, Table 1 and SupplementaryFig. 3 online). As expected, the structure of N2-Mad2 R133A closelyresembles that of the Mad1- or Cdc20-bound form of Mad2 exceptthat the ligand-binding site is unoccupied (Fig. 2). As in the ligand-bound structure of Mad2, the C-terminal region of N2-Mad2 under-goes a marked conformational rearrangement with strands β8 and β9taking the place of β1. Because N2-Mad2 has largely undergone theconformational change that occurs upon binding to Cdc20, it isexpected to be more amenable to binding Cdc20 and more potent inAPC/C inhibition than N1-Mad2. For example, for N1-Mad2 to bindCdc20, the interactions between β8 and β6 need to be disrupted first.This then allows the Mad2-binding motif of Cdc20 to gain access to β6for the formation of an antiparallel β-sheet. In the case of N2-Mad2,one edge of strand β6 is already solvent-exposed and available to formthe β-sheet interactions with the Mad2-binding motif of Cdc20.

N2-Mad2 is much more potent in APC/C inhibitionTo test whether the two conformers of Mad2 R133A indeed have dif-ferent inhibitory activities toward APC/C, we evenly divided a sampleof N1-Mad2 R133A into two portions, one of which was allowed toconvert into N2-Mad2 R133A through an 18 h incubation at 30 °C (to reach an equilibrium with 88% of the Mad2 protein being the N2conformer). N2-Mad2 R133A was much more potent in inhibitingAPC/CCdc20 and blocking the degradation of cyclin B1 in mitoticX. laevis egg extracts (Fig. 3). A sample of wild-type N1-Mad2monomer was also evenly divided: half of the sample was converted to

wild-type N2-Mad2 dimer after an 18 h incubation at 30 °C whereasthe other half was kept frozen and remained in the N1 conformation.Again, the wild-type N2-Mad2 dimer inhibited cyclin B1 degradationmore potently than did the wild-type N1-Mad2 monomer (Fig. 3b,c).Notably, the wild-type N2-Mad2 dimer was only slightly more activethan the N2-Mad2 R133A monomer (Fig. 3c). Thus, Mad2

Figure 3 N2-Mad2 more potently blocks the activity of APC/C than does N1-Mad2. (a,b) In vitro–translated 35S-labeled full-length human cyclin B1,a known APC/C substrate, was added to mitotic X. laevis egg extracts in thepresence of various forms of recombinant purified Mad2. Final concentrationof each Mad2 protein, 20 µM. In experiments involving the addition of twodifferent forms of Mad2 proteins, the two forms of Mad2 were added at amonomer molar ratio of 1:1. Samples were taken at the indicated timepointsand analyzed by SDS-PAGE followed by autoradiography. These assays weredone at room temperature. Given the short duration of the assays and thehalf-life of N1-Mad2 (see Fig. 6b), the amount of N1-Mad2 that hadtransformed into N2-Mad2 during the assays was negligible. The half-life of cyclin B1 in each experiment is indicated. WT, wild type. (c) Conditionswere the same as in a,b except that the degradation assay was carried out for 30 min and with varying concentrations of different Mad2 proteins. Thecyclin B1 protein remaining in the assay after 30 min was quantified andplotted against the concentration of Mad2.

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dimerization is not required for efficient inhibition of APC/C and N2-Mad2 is probably the activated form of Mad2.

We have reported previously that a Mad2 mutant with its C-terminal ten residues deleted (∆C-Mad2) forms monomers exclusively and does not interact with Cdc20 (refs. 16,19). Asexpected, ∆C-Mad2 exists as the N1 conformer and loses its ability toundergo the N1-N2 transition16. Consistent with earlier find-ings19,20, both ∆C-Mad2 and the wild-type N1-Mad2 monomer hada dominant-negative effect on the APC/C-inhibitory activity of thewild-type N2-Mad2 dimer (Fig. 3b). Notably, N1-Mad2 R133A didnot block the function of N2-Mad2 R133A in a dominant-negativefashion (Fig. 3b).

N1-Mad2 heterodimerizes with N2-Mad2The dominant-negative effect of ∆C-Mad2 in X. laevis egg extracts wasparticularly puzzling, as dominant-negative mutants typically achievetheir effect by sequestering proteins involved in the same pathway.However, ∆C-Mad2 failed to interact with all known Mad2-bindingproteins, including Mad1, Cdc20 and Cmt2 (data not shown). BecauseN1-Mad2 R133A had lost this dominant-negative function, we there-fore considered that ∆C-Mad2 and wild-type N1-Mad2 could formheterodimers with wild-type N2-Mad2 and that the conformationallymixed N1–N2 heterodimers were inactive for APC/C inhibition. Basedon gel filtration chromatography, ∆C-Mad2 indeed formed a stableheterodimer with wild-type N2-Mad2, but did not interact with wild-type N1-Mad2 (Fig. 4a). Furthermore, the ∆C-Mad2 R133A mutantdid not form a heterodimer with wild-type N2-Mad2 (Fig. 4a). As pre-dicted, ∆C-Mad2 R133A did not block the function of wild-type N2-Mad2 in X. laevis extracts (data not shown). Wild-type N1-Mad2also transiently formed heterodimers with wild-type N2-Mad2 basedon the 1H-15N HSQC spectrum of the 1:1 mixture of the two conform-ers (data not shown). However, unlike the wild-type ∆C-Mad2–Mad2heterodimers, the wild-type Mad2 N1–N2 heterodimer was unstableand converted to the wild-type N2-Mad2 homodimer. Taken together,these results suggest that dimerization between the N1 and N2 con-formers of Mad2 might cause the dominant-negative effects of ∆C-Mad2 and the wild-type N1-Mad2 monomer. The R133A muta-tion might disrupt the formation of N1–N2 heterodimers, thus possi-bly explaining the inability of N1-Mad2 R133A and ∆C-Mad2 R133Ato block the function of N2-Mad2 in vitro.

∆C-Mad2 blocks the spindle checkpoint in vivoOur results suggest a model whereby the N1-N2 structural transitionof Mad2 constitutes a key step in spindle checkpoint signaling. Wenext sought to obtain in vivo data supporting this model and the bio-logical relevance of both forms of Mad2. For this purpose, we analyzedlysates from HeLa cells using coimmunoprecipitations among Mad1,Mad2 and Cdc20, and monitoring their fractionation patterns on gelfiltration and anion exchange columns. In HeLa cells arrested at theG1-S boundary by thymidine and with an inactive spindle checkpoint,∼ 25% of Mad2 was associated with Mad1 and 75% was free; upontreatment with nocodazole, a microtubule-depolymerizing drug, toactivate the spindle checkpoint, 20% of Mad2 remained bound toMad1, 50% formed complexes with Cdc20 and only 30% of Mad2 wasfree (data not shown). These results are consistent with previouslypublished data5–7. The Mad1–Mad2 complex and Mad2–Cdc20-containing complexes have been extensively characterized5–7, andstructural studies have shown that they involve the N2 conformationof Mad211,15. Hence, in subsequent experiments we attempted to gaininsight into the conformation of ‘free’ Mad2 in HeLa cells.

Isolation of free Mad2 from the thymidine- and nocodazole-arrested HeLa cells by gel filtration on a Superdex-75 column showedonly monomeric Mad2 and no Mad2 dimer (Fig. 4b). The monomericpools of Mad2 were further fractionated on a Mono-Q anion-exchange column. Endogenous Mad2 from both thymidine- andnocodazole-arrested HeLa cells eluted at the same salt concentrationas the purified wild-type N1-Mad2 (Fig. 4c). This result suggests that,despite the fact that N1-Mad2 is less stable than N2-Mad2 thermo-dynamically in vitro, N1-Mad2 might be the predominant free Mad2species in human cells, regardless of the status of the spindle check-point. In addition, Mad2 that is produced in Sf9 insect cells, whose cellular environment is more similar to that of mammalian cells thanto that of bacteria21, predominantly exhibits the N1 conformation (see Fig. 6a below).

Figure 4 Endogenous Mad2 protein from HeLa cells exhibits the N1conformation. (a) ∆C-Mad2 forms a heterodimer with wild-type N2-Mad2(N2-Mad2 WT), but not with N1-Mad2 WT. In contrast, ∆C-Mad2 R133Adoes not interact with either form of Mad2 WT. The indicated pairs of Mad2proteins were mixed and fractionated on a Superdex-75 column. Thepositions of the Mad2 dimer and monomer are indicated. (b) Lysates of HeLacells treated with thymidine (Thy) or nocodazole (Noc) were fractionated ona Superdex-75 column and blotted with anti-Mad2. The positions of theMad2 dimer and monomer are indicated. (c) Purified recombinant N1-Mad2WT and N2-Mad2 WT were fractionated on a Mono-Q column. The fractionswere analyzed by SDS-PAGE followed by Coomassie staining (the top twopanels). Note that the dimeric N2-Mad2 WT fractionated broadly on the Q column due to minor populations of monomeric N1-Mad2 WT and the N1-N2 Mad2 WT conformationally mixed heterodimers. Fraction 22 of the HeLa lysates from the Superdex-75 column described in (b) was further fractionated on the Mono-Q column. The fractions were blotted with anti-Mad2 (the bottom two panels).

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transfected cells (Supplementary Fig. 4 online). For comparison, wealso depleted Mad2 from HeLa cells using RNA interference (RNAi).The Mad2 protein was efficiently depleted in HeLa cells transfectedwith Mad2-specific siRNA (Fig. 5b). About 19% of Mad2 RNAi cellsarrested in mitosis in the presence of nocodazole (Fig. 5c,d). Theseresults show that, in HeLa cells, the detrimental effect of ∆C-Mad2overexpression on the spindle checkpoint is comparable to that causedby depleting Mad2 with RNAi.

Because ∆C-Mad2 heterodimerizes with N2-Mad2 and does notbind to any of the known Mad2-binding proteins, ∆C-Mad2 mostlikely blocks the spindle checkpoint by sequestering N2-Mad2 in livingcells. Notably, overexpression of ∆C-Mad2 R133A in HeLa cells didnot substantially alter the checkpoint-mediated mitotic arrest in thepresence of nocodazole (Fig. 5a,d), indicating that ∆C-Mad2 R133Adoes not interfere with checkpoint signaling in vivo. Hence, a single

We did not detect the formation of either monomeric or dimericfree N2-Mad2 in HeLa cell lysates. This was not surprising becauseN2-Mad2 might quickly associate with Cdc20 and/or other factorssuch as Cmt2, causing a failure to detect this form of free Mad2 inHeLa cells. In addition, there might be yet unidentified mechanisms inHeLa cells that actively convert N2-Mad2 to N1-Mad2. Because ∆C-Mad2 selectively binds to N2-Mad2 but does not bind to N1-Mad2, ∆C-Mad2 serves as a ‘sensor’ of N2-Mad2. To address whether N2-Mad2 isrelevant in vivo, we next examined the pheno-types of HeLa cells transfected with plasmidsencoding wild-type Mad2, Mad2 R133A, ∆C-Mad2 and ∆C-Mad2 R133A (Fig. 5).Overexpression of Mad2 R133A arrestedHeLa cells at metaphase as efficiently as wild-type Mad2 in the absence of spindle-damag-ing agents owing to the untimely formation ofthe Mad2–Cdc20 complex (Fig. 5). This againconfirmed that dimerization of Mad2 was notrequired for its function. On the other hand,overexpression of ∆C-Mad2 and ∆C-Mad2R133A had no effect on the cell cycle in theabsence of spindle damage (Fig. 5a,d), consis-tent with the fact that both mutants failed tointeract with Cdc20 (Supplementary Fig. 4online). As described above, ∆C-Mad2, butnot ∆C-Mad2 R133A, selectively forms het-erodimers with N2-Mad2 and inhibits itsfunction in X. laevis egg extracts. Consistentwith these results, cells expressing ∆C-Mad2failed to accumulate in mitosis in the presenceof nocodazole, indicating that cells expressing∆C-Mad2 contained a defective spindle check-point (Fig. 5a,d). As expected, ∆C-Mad2 didnot interact with Mad1 or Cdc20 in these

Figure 5 Overexpression of ∆C-Mad2 partially blocks spindle checkpointsignaling in living cells. (a) HeLa cells were transfected with pCS2 vectorsencoding the indicated Mad2 proteins together with pCS2-GFP. Cellstransfected with pCS2-GFP alone were also included as controls. The cellswere either untreated (top) or treated with 300 nM nocodazole (bottom)24 h post-transfection. After another 18 h, cells were incubated withHoechst 33342 and imaged with a Zeiss Axiovert 200 M microscope. GFP is green and DNA is pseudo-colored blue. Mitotic cells are round and contain condensed DNA. (b) HeLa cells either mock transfected or transfected with Mad2 siRNA were dissolved in SDS sample buffer,separated on SDS-PAGE and blotted with the indicated antibodies. APC2 was used as a loading control. (c) HeLa cells mock transfected ortransfected with Mad2 siRNA were treated with nocodazole (300 nM) for 16 h, incubated with Hoechst 33342 and directly visualized using aninverted fluorescence microscope. Differential interference contrast (DIC)image is gray and DNA is pseudo-colored blue. (d) The mitotic indices of the transfected cells in a,c were quantified. At least 400 cells werecounted for each transfection. WT, wild type.

Figure 6 Mad1 facilitates the N1-N2 conversionof Mad2 in vitro. (a) The high-field region of 1D 1H spectra of various forms of Mad2. TheVal197 methyl peaks (–0.35 p.p.m.) used tomonitor the appearance of N2-Mad2 areindicated by arrows. (b) The N1-N2 conversion of Mad2 R133A alone (�), Mad2 R133A with

the addition of 1:0.25 molar ratio of a control Mad1 peptide (residues 485–504) that does not bind toMad2 (● ), or Mad2 R133A in the presence of 1:0.25 molar ratio of the Mad2-binding peptide of Mad1 (residues 540–551) (● ) monitored by 1D 1H experiments at 30 °C. The relative intensity of themethyl peak of Val197 (–0.35 p.p.m.) is plotted against time. Assuming two-state kinetic behavior, thekinetic curves were fitted with DYNAFIT42 with the following models and parameters: for Mad2 R133Aalone, N1 N2 (k1 / k–1), k1 = 3.0 × 10–5 s–1, k–1 = 5.2 × 10–6 s–1; for Mad2 R133A with Mad1, N1 + Mad1 N2–Mad1 (k1 / k–1), N2–Mad1 N2 + Mad1 (k2 / k–2), k1 = 4.0 × 103 M–1 s–1, k–1 = 1.5 × 10–2 s–1, k2 = 2.8 × 10–4 s–1, k–2 = 23 M–1 s–1.

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point mutation (R133A) known to disrupt the heterodimerization of∆C-Mad2 and N2-Mad2 also abrogates the checkpoint-inhibitoryeffect of ∆C-Mad2. These results strongly suggest that the dominant-negative effect of ∆C-Mad2 in HeLa cells is due to the sequestration ofthe N2 conformer of Mad2. Though unlikely, ∆C-Mad2 may exert itseffect by binding to a yet unidentified spindle checkpoint protein andthe R133A mutation may disrupt the interaction between ∆C-Mad2and this putative factor. In addition, it is unclear whether ∆C-Mad2sequesters free N2-Mad2, Mad1-bound Mad2, or Cdc20-boundMad2. Despite these caveats and the necessarily indirect nature of ourexperiments, our results suggest that N2-Mad2 is involved in spindlecheckpoint signaling in vivo.

Mad1 facilitates the N1-N2 conversion of Mad2 in vitroWe do not know whether and how an active spindle checkpoint stimu-lates the formation of the active N2-Mad2 conformer. However, as

predicted by our initial hypothesis, Mad2 dissociated from theMad1–Mad2 complex has the N2 conformation, based on the patternof its NMR spectra (Supplementary Fig. 5 online). This suggests thatMad1 might have a role in the formation of N2-Mad2. To explorewhether Mad1 can accelerate the formation of N2-Mad2 in vitro, wenext investigated the rate of conversion from N1-Mad2 R133A to N2-Mad2 R133A in the presence or absence of a Mad2-binding pep-tide of human Mad1 (residues 540–551). A methyl group of Val197 hasa unique 1H chemical shift at –0.35 p.p.m. in the N2 form of Mad2R133A, but not in the N1 form (Fig. 6a). The 1D spectrum of the wild-type Mad2 dimer also contained the Val197 methyl signal at–0.35 p.p.m., albeit with a much broader linewidth, again suggestingthat the Mad2 dimer is in the N2 conformation (Fig. 6a). We moni-tored the intensity of the Val197 methyl peak in a series of 1D experi-ments at 30 °C over a period of 24 h to determine the rate of formationof the N2 conformer (Fig. 6b). N1-Mad2 R133A spontaneously con-verted to N2-Mad2 R133A with a forward reaction rate of 3.0 × 10–5 s–1

(equivalent to a lifetime (defined as 1 / k) of 9.4 h for N1-Mad2 R133A)and a backward reaction rate of 5.2 × 10–6 s–1. The extremely slow rateof the reaction indicated a large energetic barrier for this structuraltransition (the activation free energy is >10 kcal mol–1). The conver-sion from N1-Mad2 R133A to N2-Mad2 R133A was greatly acceler-

ated (∼ 9.3-fold) with the addition of a substoichiometric amount ofthe Mad2-binding Mad1 peptide (a Mad1/Mad2 ratio of 1:4, similar tothat present in HeLa cells) whereas a control peptide had little effect(Fig. 6b). The kinetics of the conversion from wild-type N1-Mad2 towild-type N2-Mad2 was more complex because of the involvement ofdimerization events, but was also accelerated by the Mad2-bindingdomain of Mad1 (data not shown). These results suggest that Mad1facilitates the N1-N2 conformational rearrangement of Mad2.

DISCUSSIONThe two-state behavior of Mad2Many examples of conformational plasticity of proteins have beenreported. Several proteins undergo marked structural changes uponcovalent modification, binding of ligands (such as the SNT PTBdomain22), or changes in buffer conditions (such as hemagglutinin23).However, in these cases, only one of the conformers is substantiallypopulated at any given condition. Ligand binding or changes in condi-tions might trigger the conformational changes by selectively stabiliz-ing the sparsely populated, but pre-existing, high-energy state. Incontrast, both structural states of Mad2 are substantially populated inthe absence of ligand binding or changes in conditions.

There are also precedents for protein conformational flexibilitywithout covalent modification or binding of cofactors. Examplesinclude α-lytic protease without the pro-region24, several domain-swapped dimers25,26, the serpins27, the bacterial signaling proteinNtrC28, the SPE7 monoclonal antibody29 and the prion-like pro-teins30. The structural plasticity of Mad2 also differs markedly fromthe conformational diversity of all these cases. In the case of α-lyticprotease24, one of the conformers is a molten-globule state and the twostates do not interconvert in the absence of the pro-region. In the casesof cyanovirin, p13suc1 and other domain-swapped dimers25,26, thetwo folded states involve dimerization and, more importantly, thedomain-swapped dimer has the same folding topology as that of themonomer. Some serpins exist in a kinetically trapped active conforma-tion and in an inactive latent conformation without covalent modifi-cation31. However, unlike Mad2, the active serpin conformation is thehigh-energy state and is not substantially populated at equilibriumbecause the latent form (with one of the loops inserted as a centralstrand in a large β-sheet) is at least 10 kcal mol–1 more stable. BothNtrC and SPE7 have multiple conformers at equilibrium28,29.However, these conformers contain the same secondary structural ele-ments and folding topology. In addition, the lifetimes of these con-formers are in the range of microseconds to tens of milliseconds28,29.In the case of prions, the two conformational states are typically not atequilibrium, and one of the two states catalyzes its own formation30.The N1-N2 conversion of Mad2 R133A follows first-order reactionkinetics, and addition of a small amount of wild-type N2-Mad2 dimerto wild-type N1-Mad2 does not appreciably alter the N1-N2 conver-sion rate of wild-type Mad2, ruling out self-propagation of the N2form of Mad2. Independently of the similarities and differences withother systems, the properties of Mad2 provide a clear example of how the conformational malleability of a protein might be used as asignaling mechanism for a fundamental biological process.

In vivo relevance of the two-state behavior of Mad2We also obtained evidence to suggest the in vivo relevance of the N1and N2 forms of Mad2. Our results are consistent with a speculativemodel whereby the N2 form of Mad2 constitutes a conformational sig-nal that activates the spindle checkpoint and Mad1 has a chaperone-like activity that accelerates the N1-N2 transition of Mad2, amongother mechanisms that might regulate this transition. This model

Table 1 Structural statistics for human N2-Mad2 R133A

Average r.m.s. deviations from experimental restraintsa (2,866 total)

NOE distance restraints

All (2,323) 0.018 ± 0.003

Hydrogen bond restraints (204) 0.021 ± 0.003

Dihedral angle restraints (339) 0.413 ± 0.007

Average r.m.s. deviations from idealized covalent geometryb,c

Bonds (Å) (3,350) 0.00216 ± 0.00004

Angles (°) (6,074) 0.413 ± 0.007

Impropers (°) (1,703) 0.308 ± 0.012

Average r.m.s. deviations of the 25 atomic coordinates (Å)

Backboned 0.56 ± 0.08

Heavy atomsd 1.06 ± 0.08

Backbonee 0.45 ± 0.08

Heavy atomse 0.95 ± 0.06

aValues in parentheses are the number of experimental restraints. bIdealizedgeometries based on CNS38 parameters (protein-allhdg.param). cValues in parenthesesindicate the total numbers of bonds, angles and impropers in the protein, respectively.dExcluding residues 106–118 and 159–176. eCalculated for residues in secondarystructure elements.

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explains the apparent paradox raised by the observations that Mad1 isrequired for binding of Mad2 to Cdc20 in vivo and that a complete lossof Mad1 function disrupts checkpoint signaling11, yet Mad1 acts as acompetitive inhibitor of Cdc20-binding by Mad2 and overexpressionof Mad1 inactivates the checkpoint11,12. Thus, the ratio of Mad1/Mad2(∼ 1:4 in HeLa cells; Z.T. & H.Y., unpublished results) seems crucial forproper spindle checkpoint signaling in vivo and may be optimal for thechaperone-like role of Mad1 proposed in our model. In the absence ofMad1, N2-Mad2 does not form efficiently, resulting in a defectivespindle checkpoint. Overexpression of Mad1 also inactivates thecheckpoint because an excess amount of Mad1 competes with Cdc20for Mad2 binding.

Several issues remain unresolved in this model. First, based on theprofile of chromatography, we showed that, surprisingly, the majorityof the monomeric pool of Mad2 in HeLa cells might be in the N1 state,which is less stable in vitro. In addition, the human Mad2 proteinexpressed in Sf9 insect cells exists predominantly in the N1 state. Atpresent, we do not know the underlying mechanism that keeps Mad2in this high-energy state in Sf9 and possibly HeLa cells. However, wespeculate that a yet unidentified ATP-dependent mechanism mightactively convert N2-Mad2 to N1-Mad2. This might help to explain ourfailure to detect free N2-Mad2 directly. Second, why is the endogenouspool of free N1-Mad2 in checkpoint-active cells unable to block thefunction of N2-Mad2? It is possible that the binding affinity betweenN1-Mad2 and N2-Mad2 is low. In X. laevis egg extracts, we used20 µM of each of the Mad2 proteins. However, the concentration ofthe endogenous Mad2 in HeLa cells is 100–300 nM, which presumablydoes not allow the efficient formation of the N1-N2 Mad2 hetero-dimer. In the case of overexpression of the wild-type Mad2, we do notknow whether the majority of the overexpressed Mad2 protein existsin N1 or N2 states. Obviously, more direct methods for detecting N1-Mad2 and N2-Mad2 are needed to address these issues. Third, thein vitro rate of N1-N2 conversion of Mad2 assisted by the Mad1 peptide is still too slow to account for the short half-life (26 s) reportedfor Mad2 at the unattached kinetochores in living cells32. Therefore,the physiological relevance of this in vitro finding is unclear at present.In the future, it will be valuable to investigate whether checkpoint-mediated phosphorylation of Mad1 or association with other proteinsmight further enhance the rate of Mad2 turnover on Mad1 in vitro andin vivo33,34.

METHODSProtein expression and purification. The coding regions of wild-type Mad2and ∆C-Mad2 were cloned into the BamHI-HindIII sites of pQE30 (Qiagen)with 5′-primers encoding a TEV protease cleavage site. The pQE30-Mad2R133A and pQE30-∆C-Mad2 R133A vectors were constructed with theQuikChange kit (Stratagene). These vectors were expressed in bacteria withinduction at 16 °C and the resulting His6-tagged fusion proteins were isolatedwith Ni2+-NTA beads (Qiagen). After the removal of the His6-tag by TEV diges-tion, the Mad2 proteins were purified with a Mono-Q column (Amersham).The N1 and N2 forms of Mad2 proteins were pooled separately and furtherpurified by a Superdex-75 column (Amersham). The TEV-cleaved recombi-nant Mad2 proteins contained only an extra glycine at their N termini.

NMR spectroscopy and structure determination. NMR experiments weredone at 30 °C on Varian Inova 500 or 600 MHz spectrometers equipped withfour channels and pulsed-field gradients. Samples contained 0.5 mM N2-Mad2R133A protein in the NMR buffer (50 mM phosphate, pH 6.8, 300 mM KCland 1 mM DTT). Sequential assignment of the backbone and side chain reso-nances was done as described16,35,36. NOEs were identified from 3D 15N or 13CNOESY-HSQC spectra. The distance restraints were derived from NOEs andhydrogen bonds as described16. Backbone φ and ψ torsional angle restraintswere derived using TALOS37. Side chain χ1 angle restraints were assigned based

on intraresidue NOE patterns. Structures were calculated with CNS38. To mon-itor the conformational change of Mad2, a series of 1D NMR spectra, each last-ing 30 min, was acquired on samples containing 0.1 mM N1-Mad2 R133A orwild-type N1-Mad2 in the NMR buffer.

Cyclin degradation assay. Cyclin degradation assays using X. laevis egg extractswere done as described19 except that in vitro–translated 35S-labeled full-lengthhuman cyclin B1 was used as the substrate instead of the 125I-labeled N-terminal fragment of X. laevis cyclin B1.

Mammalian cell culture, transfection and microscopy. HeLa cells were grownin DMEM (Invitrogen) supplemented with 10% (v/v) FBS. To arrest cells at theG1-S boundary, 2 mM thymidine was added to the medium for 18 h. To obtaincells arrested at metaphase with an active spindle checkpoint, cells were treatedwith 300 nM nocodazole for 18 h. For the fractionation of endogenous Mad2,cells were incubated with the lysis buffer (50 mM Tris-HCl, pH 7.7, 100 mMKCl, 1 mM DTT, 0.5 µM okadaic acid and protease inhibitors) and passedthrough a 21-gauge needle several times. The cleared supernatant was thenloaded onto a Superdex-75 column.

Transfection of HeLa cells with pCS2 plasmids encoding wild-type Mad2,Mad2 R133A, ∆C-Mad2 and ∆C-Mad2 R133A was done using the Effectenereagent (Qiagen) according to the manufacturer’s protocol; pCS2-GFP wascotransfected into these cells to provide a fluorescent marker. After 36 h, cellswere treated with 300 nM nocodazole for 18 h, stained with Hoechst 33342, anddirectly observed with a Zeiss inverted fluorescence microscope. The siRNAoligonucleotides targeting Mad2 were chemically synthesized at an in-housefacility, and contained sequences corresponding to nucleotides 143–165 of thehuman Mad2 coding region. The annealing of the siRNAs and subsequenttransfection of the RNA duplexes into HeLa cells were done using theOligofectamine reagent (Invitrogen) exactly as described39.

Coordinates. The atomic coordinates of N2-Mad2 R133A have been depositedin the Protein Data Bank (accession code 1S2H).

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSWe thank Y. Liu for reading the manuscript critically and R. Hampton forsuggestions. This work was supported by the American Cancer Society (to X.L.),Association pour la Recherche contre le Cancer (ARC) (to K.W.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (COE Research) (to T.M.), the Welch Foundation (to J.R. and H.Y.), the Packard Foundation (to H.Y.), the Burroughs Wellcome Fund (to H.Y.), the W.M. Keck Foundation (to H.Y.) and the US National Institutes of Health (to H.Y.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 13 November 2003; accepted 23 February 2004Published online at http://www.nature.com/natstructmolbiol/

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