T H E J O U R N A L O F C E L L B I O L O G Y
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J. Cell Biol. Vol. 183 No. 5 761–768
Correspondence to S. Walter Englander: firstname.lastname@example.org; or Ben
E. Black: email@example.com
Abbreviations used in this paper: APC, anaphase-promoting complex/cyclo-
some; C-Mad2, closed Mad2; I-Mad2, intermediate Mad2; MBP1, Mad2-binding
peptide 1; MCC, mitotic checkpoint complex; O-Mad2, open Mad2.
The essential goal of mitosis is the equal distribution of sister
chromatids into genetically identical daughter cells ( Cleveland
et al., 2003 ; Walczak and Heald, 2008 ). Chromosome segrega-
tion is directed by the centromere, a locus epigenetically de-
fi ned by a specialized chromatin domain marked by nucleosomes
in which the histone variant CENP-A (centromere protein A)
replaces H3 ( Black and Bassett, 2008 ). The kinetochore, an
enormous protein assembly consisting of > 80 known proteins,
assembles upon the centromere of each chromatid and connects
to microtubule-based fi bers that extend from opposite poles of
the mitotic spindle. Accurate kinetochore attachment to the
spindle is monitored by a diffusible checkpoint signal termed
the mitotic checkpoint (also referred to as the spindle assembly
checkpoint; Musacchio and Salmon, 2007 ; Yu, 2007 ). The
checkpoint inhibits mitosis, halting progression to anaphase un-
til all chromosomes are aligned on the metaphase plate and
every kinetochore is properly attached to the spindle ( Fig. 1 A ).
On and off switching of the mitotic checkpoint must be
fast and defi nitive because either a weak checkpoint or an asyn-
chronous metaphase to anaphase transition leads to irreversible
missegregation of one or more chromosomes. The checkpoint
must be active upon entry into mitosis and suffi ciently robust so
that checkpoint activation is maintained if even a single kineto-
chore remains unattached to the spindle ( Fig. 1 B ). After proper
spindle attachment to all kinetochores, the checkpoint rapidly
inactivates to allow for the destruction of mitotic targets (e.g.,
cyclin B and securin), which leads to synchronous chromosome
separation and segregation. Inappropriate early inactivation of
the checkpoint produces lethal chromosomal missegregation
( Kops et al., 2004 ; Michel et al., 2004 ). However, a functional
mitotic checkpoint is required for tumor cell death resulting
from treatment with microtubule toxins such as taxol that are
widely used in the clinic ( Gascoigne and Taylor, 2008 ).
The Mad2 protein is a centrally important regulator of the
mitotic checkpoint machinery. Its activity is controlled by switch-
ing between its two different native conformations, open Mad2
(O-Mad2; also referred to as Mad2 N1 ; Fig. 1 C ) and closed Mad2
(C-Mad2; also referrred to as Mad2 N2 ; Fig. 1 D ; Luo et al., 2004 ;
De Antoni et al., 2005 ). Before checkpoint activation, freely dif-
fusible monomeric Mad2 is thought to exist largely as O-Mad2,
its inactive conformation, as is common for many regulatory pro-
teins. Conformational conversion from inactive free O-Mad2 to
active free C-Mad2 is catalyzed by a self – self interaction, namely
by binding to the C-Mad2 subunit of a Mad1 – C-Mad2 complex
( Luo et al., 2000 ; Sironi et al., 2002 ; Vink et al., 2006 ) anchored
at kinetochores that are not yet properly engaged with a spindle
( Fig. 1 E ; Chen et al., 1998 , 1999 ; Waters et al., 1998 ). Although
a direct physical demonstration that Mad2 structural conversion
is catalyzed by unattached kinetochores is currently lacking, puri-
fi ed Mad1-bound Mad2 is known to catalyze the O-Mad2 →
C-Mad2 transition in the absence of any other effector molecules
( Yang et al., 2008 ). Newly converted Mad2 releases from the ki-
netochore and blocks premature progression to anaphase by bind-
ing to and deactivating Cdc20 in conjunction with other essential
checkpoint proteins (including BubR1 kinase and Bub3) as part
of a mitotic checkpoint complex (MCC; Fig. 1 E ; Hardwick et al.,
2000 ; Sudakin et al., 2001 ). Although the checkpoint remains ac-
tive, the inhibition of Cdc20 by C-Mad2 serves to restrain an E3
ubiquitin ligase known as the anaphase-promoting complex/
cyclosome (APC; Fig. 1 E ; Musacchio and Salmon, 2007 ; Yu, 2007 ).
The metamorphic Mad2 protein acts as a molecular
switch in the checkpoint mechanism that monitors proper
chromosome attachment to spindle microtubules during
cell division. The remarkably slow spontaneous rate of
Mad2 switching between its checkpoint inactive and ac-
tive forms is catalyzed onto a physiologically relevant
time scale by a self – self interaction between its two forms,
culminating in a large pool of active Mad2. Recent struc-
tural, biochemical, and cell biological advances suggest
that the catalyzed conversion of Mad2 requires a major
structural rearrangement that transits through a partially
The Mad2 partial unfolding model: regulating
mitosis through Mad2 conformational switching
John J. Skinner , Stacey Wood , James Shorter , S. Walter Englander , and Ben E. Black
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104
© 2008 Skinner et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 183 • NUMBER 5 • 2008 762
Mad2 differs strikingly from most regulatory proteins.
Other proteins that change structure drastically, known as meta-
morphic proteins ( Murzin, 2008 ), require the selective stabiliza-
tion of their intrinsically less stable active form through substrate
binding, chemical modifi cation, or environmental change. For
Mad2, the structural changes from the inactive form to the active
Once all kinetochores have properly attached to the spindle,
Mad2 deactivates and releases Cdc20, allowing it to bind and ac-
tivate the APC. APC–Cdc20 ubiquitinates several key mitotic
substrates, including securin and cyclin B, leading to their re-
moval by the proteasome and initiation of the metaphase to ana-
Figure 1. The mitotic checkpoint ensures equal partitioning of chromosomes in anaphase. (A) A human tissue culture cell progressing through mitosis with
time indicated in minutes. In the top row, chromosomes (green) are overlaid with a differential interference contrast image of the entire cell. Sister chroma-
tids align at the metaphase plate early in mitosis and wait for ? 20 min before chromatid separation in anaphase. Upon fi nal chromosome alignment, the
mitotic checkpoint signal decays, allowing the cell to enter anaphase and initiate simultaneous separation of sister chromatids. (B) The mitotic checkpoint
signal, comprised in part by a diffusible pool of C-Mad2, emanates from kinetochores that have not yet properly engaged the microtubule-based spindle.
A single unattached chromosome is suffi cient to generate a checkpoint signal that arrests mitosis before anaphase. (C and D) Interconversion between
inactive O-Mad2 (PDB 1DUJ ; Luo et al., 2000 ) and checkpoint-active C-Mad2 (PDB 1S2H ; Luo et al., 2004 ) involves a major secondary and tertiary
structural reorganization of N-terminal (blue) and C-terminal (red) segments. (E) Unattached kinetochores contain the checkpoint protein Mad1, which
recruits C-Mad2, providing a catalytic surface for the conversion of the soluble pool of inactive O-Mad2 to active C-Mad2. C-Mad2 is able to bind and
inhibit Cdc20 within the MCC, halting progression to anaphase. The Cdc20 – C-Mad2 complex may also act to catalyze conversion of the O-Mad2 pool,
although this aspect of Mad2 signaling remains controversial ( Yu, 2006 ; Musacchio and Salmon, 2007 ). (B and E) Chromosomes are drawn in green with
their kinetochores drawn in red.
763UNFOLDING AND REFOLDING OF MAD2 TO REGULATE MITOSIS • Skinner et al.
form are unusually large ( Fig. 1, C and D ) and remarkably slow
( Luo et al., 2004 ). Furthermore, Mad2 is found initially out of
equilibrium in its inactive form (O-Mad2) even though its active
form (C-Mad2) is the more stable conformation ( Luo et al.,
2004 ). Thus, checkpoint activation simply requires Mad2 to
reach its equilibrium distribution. These properties raise key
questions about the mechanism of mitotic checkpoint regula-
tion. How do effector molecules modulate the rate of Mad2 in-
terconversion? Could Mad2 regulation involve kinetic trapping
in one of its two conformational states? Do transient conforma-
tional intermediates play a functional role?
The Mad2 structural rearrangement
The spontaneous Mad2 activation reaction, O-Mad2 → C-Mad2,
proceeds with a lifetime of 9 h; the reverse reaction is sixfold
slower ( Luo et al., 2004 )! These unusually slow interconversion
rates stem from the magnitude of the structure change, which
involves a complete rearrangement of the secondary and tertiary
structure of 60 out of 205 amino acids. In O-Mad2, the N-terminal
segment forms a long loop and a short ? strand ( ? 1) that con-
nects to the static core ( Fig. 1 C ). In the transition to C-Mad2,
this segment loses its ? conformation and reconfi gures, adding
two more turns to the ? A helix ( Fig. 1 D ; Luo et al., 2002 , 2004 ;
Sironi et al., 2002 ). The C terminus undergoes an even more
dramatic change. In O-Mad2, the C-terminal segment forms
strands ? 7 and ? 8 (and connecting loops) that dock onto the
static core ? 6 strand. In C-Mad2, the whole segment moves to
the opposite side of the major ? sheet and forms two new
strands, ? 8 ? and ? 8 ? , with a completely different hydrogen-
bonding network. Overall, the transition to the C-Mad2 con-
former relocates the N-terminal segment to make room for the
incoming C-terminal segment, the displacement of which ex-
poses an extended active site that is occluded in O-Mad2.
The active site of Mad2 is tailored, remarkably, to interact
with both its upstream activator Mad1 ( Fig. 2 A ) and its down-
stream target Cdc20 ( Luo et al., 2002 ; Sironi et al., 2002 ).
Although Mad1 and Cdc20 appear to be otherwise unrelated,
their Mad2-interacting regions are highly homologous and can
be mimicked by a synthetic 12-residue consensus sequence pep-
tide (Mad2-binding peptide 1 [MBP1]; Fig. 2 B ; Luo et al.,
2002 ). These partners bind by incorporating into the major
Mad2 ? sheet as a single ? strand, interacting with the ? 6 strand
and a new ? 7 ? strand that forms upon the binding ( Luo et al.,
2000 , 2002 ; Sironi et al., 2002 ). As shown in Fig. 2 (A and B) ,
they actually thread through the C-Mad2 sheet like links in a
concatenated chain. Once Mad1 binds to Mad2, it forms a very
stable complex with no detectable turnover in 4 min, as detected
with purifi ed components by FRAP ( Vink et al., 2006 ), correlat-
ing with earlier cell-based FRAP measurements of the hyper-
stable pool of kinetochore-bound C-Mad2 that is presumably
bound to Mad1 ( Shah et al., 2004 ).
Figure 2. Mad2-containing complexes. (A and B) The displacement of
the C-terminal segments in C-Mad2 exposes a new ? sheet edge that can
incorporate Mad1 (A; PDB 1GO4 ; Sironi et al., 2002 ), Cdc20, or the
synthetic peptide MBP1 (B; PDB 2V64 ; Mapelli et al., 2007 ) between
newly exposed ? 6 and newly formed ? 7 ? . In the crystal structure of the
O-Mad2 – C-Mad2 dimer (B), asymmetrical dimerization occurs mainly
through the unaltered core of Mad2 (gray to tan) but also includes the ? 8 ?
strand that is unique to C-Mad2 (coloring as in Fig. 1 C ). O-Mad2, the
form undergoing conversion (tan), interacts with C-Mad2 only through its
unchanging core. (C) A reaction scheme for Mad2 catalysis. Mad2 (red)
represents the molecule undergoing conversion. Uncatalyzed Mad2 inter-
conversion proceeds far more slowly (lifetime > 9 h; Luo et al., 2004 ) than
the duration of metaphase ( ? 20 min). The Mad2 structural rearrangement
is catalyzed by binding to the Mad1 – C-Mad2 complex. In this reaction
scheme, catalysis by induced fi t would increase the forward O-Mad2 →
C-Mad2 rate, whereas the conformational selection of C-Mad2 would re-
duce the reverse O-Mad2 ← C-Mad2 rate. It is unknown whether Mad2
releases from the Mad1 – C-Mad2 dimer as fully folded C-Mad2 or as a
partially unfolded intermediate.
JCB • VOLUME 183 • NUMBER 5 • 2008 764
structure; rather, it stabilizes the selected form by decreasing
the reverse rate. Therefore, conformational selection of C-Mad2
can be effective only if conformer sampling (O-Mad2 → C-Mad2)
is appropriately rapid. If rapid conformational sampling oc-
curred naturally, catalysis would not be necessary because the
target C-Mad2 is actually the more stable form ( Luo et al., 2004 ),
ruling out conformational selection of C-Mad2 ( Fig. 2 C ).
In summary, recent structures elegantly display the static Mad2
dimerization interface, but they do not suggest a mechanism to
explain how the C-Mad2 – O-Mad2 interaction catalyzes the
O-Mad2 → C-Mad2 transition.
An unfolding model for Mad2
It is hard to envision how any kind of straightforward conforma-
tional conversion (e.g., by a hinging or rigid body motion) could
accomplish the major structural rearrangement between the two
natively folded Mad2 forms. Rather, the conformational rear-
rangement is so extensive that it seems to require a signifi cant
unfolding of Mad2 to some transient high energy intermediate
followed by kinetic partitioning between the two alternative
forms upon refolding. Similarly, the fact that the main chain of
Mad1 and Cdc20 actually threads through the major ? sheet of
C-Mad2 seems to require some transitional partially unfolded
intermediate from which the C terminus could refold around the
ligand upon binding.
A precedent for conformational change through partial
unfolding can be found in the much smaller cytochrome c al-
kaline transition. At an elevated pH, the residue ligated to
heme is switched from Met80 to the neighboring Lys79. Rather
than simply shifting over by one amino acid residue, the tran-
sition involves the unfolding and refolding of a 15-residue
loop that contains the two critical residues. The loop has been
shown to unfold and refold repeatedly under native conditions
as a cooperative unit known as a foldon. The stability of the
loop foldon determines the equilibrium between the Met80-
liganded and Lys79-liganded forms ( Maity et al., 2006 ), and
the foldon unfolding rate limits the kinetics of the transition
( Hoang et al., 2003 ). More generally, recent work indicates
that many proteins act as accretions of foldon units that repeat-
edly unfold and refold under native conditions. It now appears
that cooperative foldons can account for the unit steps in pro-
tein folding pathways, and, having reached the native state,
their continuing dynamic unfolding and refolding behavior
can be exploited to control ligand on and off rates ( Englander
et al., 2007 ) and even allosteric communication ( Hilser and
Thompson, 2007 ).
Can the emerging foldon paradigm help to explain the
Mad2 conformational switching mechanism? In addition to the
aforementioned structural issues (e.g., massive rearrangement
and threading), some other Mad2 folding – related observations
are suggestive. Chemically denatured Mad2 spontaneously re-
folds into a nonequilibrium mixture of its two alternative con-
formations (C-Mad2 and O-Mad2 in a 2:1 ratio; Luo et al.,
2004 ). The implication is that the refolding pathway contains
some intermediate stage from which Mad2 partitions into its
two different stable forms ( Fig. 3, A and B ). Spontaneous
For the sake of simplicity, it is often stated that C-Mad2
itself is competent to bind Mad1 or Cdc20. However, in this
binding reaction, Mad2 must expose a binding site, load its
binding partner, and lock it in place. This implies that binding to
either Mad1 or Cdc20 requires a substantial local rearrange-
ment of Mad2 structure ( Mapelli et al., 2007 ; Yang et al., 2008 ).
Although the possibility exists that Mad1 and Cdc20 may them-
selves unfold, thread through the Mad2-binding loop, and re-
fold, partial unfolding of Mad2 itself seems more likely, especially
because the O-Mad2 → C-Mad2 conversion appears to require a
similar partial unfolding. In this view, a partially unfolded inter-
mediate form of Mad2 would be required for Cdc20 binding
and APC inhibition.
Conformational switching models
On time scales relevant to cell biology, the great majority of
biomolecules assume their equilibrium distribution among
alternative conformations, and their rates of interconversion
can be safely ignored. However, the spontaneous O-Mad2 →
C-Mad2 conversion rate (many hours) is clearly inadequate for the
rapid checkpoint activation required to inhibit anaphase imme-
diately upon mitotic entry. The conversion of freely diffusible
O-Mad2 is catalyzed by its self-interaction with the C-Mad2
partner of the kinetochore-bound Mad1 – C-Mad2 complex
( Fig. 2 C ). How is this catalytic event accomplished? Thermody-
namic principles dictate that molecular binding partners pro-
mote structure change in allosteric proteins by binding more
strongly to the favored form. Two common structure change
models exist. Association may promote the structure change by
sacrifi cing some of its binding energy to forcefully distort the
protein conformation (induced fi t model), or selection may oc-
cur among preexisting dynamically cycling protein conforma-
tions by more strongly binding to and thereby trapping the
preferred partner (conformational selection model).
If the O-Mad2 → C-Mad2 conversion is catalyzed by in-
duced fi t, the structure of Mad2 in the catalytic complex should
display the activation mechanism. Mapelli et al. (2007) crystal-
lized a valid replica of the Mad1 – C-Mad2 – O-Mad2 catalytic
complex ( Fig. 2 B ). The O-Mad2 subunit was trapped in the
open conformation by shortening the loop connecting the ? 5
strand to the ? C helix. The O-Mad2 loopless mutant (O-Mad2 LL )
was dimerized with a C-Mad2 molecule that was bound in turn
to the synthetic activation peptide MBP1 to make a stable
MBP1 – C-Mad2 – O-Mad2 LL complex. The crystal structure of
the complex reveals that the dimerization surface of O-Mad2,
the form undergoing conformational change, only involves seg-
ments that are not substantially altered upon the O-Mad2 →
C-Mad2 switch. Thus, it does not appear that the interaction would
serve to forcefully induce the transition, providing evidence
against an induced fi t mechanism.
In the case of a conformational selection mechanism,
one can expect that the catalyzing kinetochore-bound Mad1 –
C-Mad2 complex would favor the closed form of the substrate
Mad2 molecule by binding to sites that are specifi c for C-Mad2.
In fact, the C-Mad2 – C-Mad2 complex does involve some of
those sites ( Yang et al., 2008 ). However, conformational selec-
tion alone does not increase the rate of conversion to the target
765UNFOLDING AND REFOLDING OF MAD2 TO REGULATE MITOSIS • Skinner et al.
stabilize the rate-limiting transition state relative to O-Mad2
and therefore increase the rate of O-Mad2 → C-Mad2 (it should
be noted that I-Mad2 may be but is not necessarily the same as
the intermediate that binds Mad1 and Cdc20 discussed in The
Mad2 structural rearrangement section).
Unfortunately, a Mad1 – C-Mad2 – I-Mad2 structure is not
likely to be solved by x-ray crystallography because partially
unfolded and dynamically interconverting structures are not
conducive to crystal formation. Available crystal structures
of pertinent dimers used Mad2 variants that would prevent
I-Mad2 formation. The Mad2 LL mutant used to obtain MBP1 –
C-Mad2 – O-Mad2 LL crystals prevents O-Mad2 from switching
into the C-Mad2 conformation by restricting the conformational
equilibration from this point is extremely slow. Thus, O-Mad2
is not itself a facile on-pathway precursor for generation of
C-Mad2. Rather, O-Mad2 appears to transit to C-Mad2 by back-
tracking through a partially unfolded intermediate Mad2 (I-Mad2;
Fig. 3 A ) and redistributing between O-Mad2 and C-Mad2 over
several equilibration cycles.
How can an unfolding-dependent binding model promote
the rate of the Mad2 conformational transition? As noted be-
fore, selective binding to C-Mad2 itself would not be helpful.
Instead, the Mad1 – C-Mad2 complex needs only to selectively
stabilize a partially unfolded intermediate on the O-Mad2 side
of the rate-limiting transition barrier, such as the hypothetical
I-Mad2 in Fig. 3 C . The stabilization of I-Mad2 would equally
Figure 3. Mad2 unfolding and refolding considerations. (A) Free energy reaction landscape for Mad2 interconversion through a partially folded inter-
mediate that lies on the folding pathway (created with Matlab version R2007a; The MathWorks, Inc.). When chemically denatured Mad2 is refolded, it
initially reaches a nonequilibrium O-Mad2 – C-Mad2 mixture, suggesting that the folding pathway to reach either form passes through a common intermedi-
ate and kinetically partitions rather than passing through one form on the way to the other. We suggest that the catalyzed interconversion seems likely, on
this and other grounds, to pass back through the same partially unfolded intermediate. (B) A notional structure for a Mad2-folding intermediate showing the
common (gray) and variable (colored as in Fig. 1 C ) segments. (C) Catalysis through intermediate stabilization. The conversion reaction of Mad2 is drawn
with (red dashed line) or without (black solid line) dimerization with the kinetochore-bound C-Mad2 – Mad1 complex. The measured C-Mad2/O-Mad2
equilibrium ratio is 8:1 ( Luo et al., 2004 ), indicating that C-Mad2 is ? 1 kcal/mol more stable than O-Mad2. Conformational selection of I-Mad2 would
equally stabilize I-Mad2 and the TS2 transition barrier relative to O-Mad2, effectively increasing the O-Mad2 → C-Mad2 rate even though the energy differ-
ence between I-Mad2 and TS2 remains unchanged. The dashed black line indicates the energy state of O-Mad2, the black arrow (left) indicates increasing
energy, the double-headed black arrow indicates the energy difference between O-Mad2 and TS2 without dimerization, and the doubled-headed red
arrow indicates the energy difference between O-Mad2 and TS2 with dimerization. (D) As it emerges from the ribosome, Mad2 may preferentially fold to
O-Mad2 because the last emerging C-terminal segment is required for forming C-Mad2, and protein folding is typically much faster than translation.
JCB • VOLUME 183 • NUMBER 5 • 2008 766
tance of seeded nucleation by amyloid for prion propagation
was realized, the prion fi eld had generated several models for
how proteins could self-perpetuate structural change. Curiously,
the Mad2 switch is strikingly reminiscent of one of the early
models proposed to explain prion propagation, termed template-
directed refolding, in which newly converted prion conformers
dissociate from the original template ( Fig. 4, B and C ; Prusiner,
1991 ; Aguzzi, 2004 ; Tuite and Koloteva-Levin, 2004 ). The main
difference between Mad2 and this prion model is that the disso-
ciation of equally infectious subunits would generate an explo-
sive chain reaction ( Fig. 4 B ). In contrast, the rate of O-Mad2 →
C-Mad2 conversion ( Fig. 4 C ) is limited by the size of the static
pool of kinetochore-bound Mad1 – C-Mad2 complex (and per-
haps also that of the MCC-bound Cdc20 – C-Mad2 complex;
De Antoni et al., 2005 ). That is, free C-Mad2 monomers do not
catalyze further conversion of O-Mad2 ( Yang et al., 2008 ).
Despite this important distinction, we note that Mad2 is a stun-
ning example of a protein that undergoes template-directed re-
folding as part of its adaptive cellular function ( Fig. 4 C ). It will
be important to determine what properties of the Mad1-bound
C-Mad2 conformation endow it with the ability to catalyze
O-Mad2 → C-Mad2 conversion. Earlier work on prions generated
the prediction that self-perpetuating conformational switching
may be used in various cell biological niches ( Lindquist, 1997 ).
Although distinct from the self-templating mechanism of amy-
loids, the template-driven refolding of Mad2 represents a clear
example in which this is the case.
The extensive secondary and tertiary structural reorganization
that accomplishes Mad2 conformational switching is analogous
search space of the N terminus ( Mapelli et al., 2007 ). The
L13A mutation used to obtain C-Mad2 – C-Mad2 crystals sta-
bilizes the native closed conformation so that the alternative
O-Mad2 or I-Mad2 forms would not signifi cantly populate
( Yang et al., 2008 ). The structure of I-Mad2 will have to be
studied by methods more applicable to dynamic systems.
Foldon-dependent unfolding behavior in other proteins has so
far been studied successfully, not by static crystallography but
by dynamic hydrogen exchange ( Englander et al., 2007 ) and
nuclear magnetic resonance relaxation dispersion ( Korzhnev
and Kay, 2008 ) methods.
The focus on folding models and kinetic trapping may
also explain another puzzling observation. The in vitro equilib-
rium ratio of C-Mad2/O-Mad2 is 8:1 ( Luo et al., 2004 ). Neverthe-
less, upon mitotic entry, a catalyzed conversion to the equilibrium
C-Mad2 form is necessary. Apparently, nascent Mad2 polypep-
tide emerging from the ribosome folds preferentially to O-Mad2
( Fig. 3 D ). This conclusion is supported by the observation that
recombinant Mad2 expressed in Escherichia coli exists pre-
dominantly as O-Mad2 (when kept at low temperature to mini-
mize interconversion) even though Mad2 refolded in solution
favors C-Mad2 by 2:1 ( Luo et al., 2004 ). The preferential non-
equilibrium folding to O-Mad2 may occur before fi nal ribo-
somal disengagement because the later emerging C terminus is
required to form C-Mad2 but not O-Mad2 (a Mad2 mutant with
10 C-terminal residues truncated cannot form C-Mad2; Luo
et al., 2004 ). Given that the time constant for the O-Mad2 →
C-Mad2 transition is > 9 h, newly expressed Mad2 will be kineti-
cally trapped as O-Mad2.
Protein synthesis alone appears to be suffi cient to create a
soluble pool of O-Mad2 poised for activation upon mitotic entry
and interaction with the kinetochore-bound Mad1 – C-Mad2
complex. It remains unknown whether or not nuclear pore-
tethered Mad1 – C-Mad2 in interphase ( Campbell et al., 2001 )
and/or spindle pole – tethered Mad1 – C-Mad2 after anaphase
onset ( Shah et al., 2004 ) are capable of catalyzing the O-Mad2 →
C-Mad2 conversion. In all of the likely models, Mad1 – C-Mad2
at the kinetochore serves as an active catalytic platform for Mad2
conversion, providing a mechanism for rapid checkpoint activa-
tion at the onset of mitosis ( De Antoni et al., 2005 ; Yu, 2006 ).
Comparison of autocatalyzed Mad2
conversion with prion propagation
The O-Mad2 → C-Mad2 conversion process has been referred to
as prionlike ( Mapelli et al., 2006 ) because it shares with prions
the general property of structural conversion of one folded state
to another via self – self interactions. Prions are proteins that can
switch to self-perpetuating infectious conformations. The pri-
ons that have been extensively characterized to date, including
PrP, Sup35, Ure2, HET-s, and Rnq1 ( Shorter and Lindquist,
2005 ), are propagated as self-templating cross ? -amyloid forms
in a reaction, wherein the equilibrium between the native and
prion states is dramatically shifted by interaction with the sta-
ble self-templating amyloid ( Fig. 4 A ). Obviously, O-Mad2 →
C-Mad2 conversion has little in common structurally with amy-
loid. For instance, amyloids do not release newly converted
monomers ( Carulla et al., 2005 ). However, before the impor-
Figure 4. Comparing the Mad2 conformational switching reaction to
models for prion propagation. (A and B) Models for prion propagation
adapted from Aguzzi (2004) . (C) A simplifi ed reaction scheme for Mad2
conversion catalyzed by self – self interactions.
767 UNFOLDING AND REFOLDING OF MAD2 TO REGULATE MITOSIS • Skinner et al.
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to the major structural rearrangements that have now been seen
for some other so-called metamorphic proteins ( Murzin, 2008 )
but is distinguished structurally, kinetically, and in its self – self
catalytic nature from any other known regulatory transition.
In this review, we suggest a foldon-dependent molecular switch-
ing mechanism in which catalytic C-Mad2 selectively binds to a
partially unfolded partner called I-Mad2. Rather than directly
stabilizing the active conformation, the binding energy promotes
equilibration that favors the more stable C-Mad2 form. The
binding catalyzes the O-Mad2 → C-Mad2 conversion by equiva-
lently lowering the energy level of the transition state. This view
relates Mad2 regulatory structure change to the foldon paradigm
that emphasizes the role of the naturally occurring folding and
unfolding behavior of nativelike foldon units in protein folding
and function ( Englander et al., 2007 ). Coherently, the same
folding/unfolding picture can explain why newly synthesized
Mad2 initially folds to and becomes trapped in a nonequilibrium
conformational distribution, and it further suggests a mecha-
nism that allows the tightly folded native C-Mad2 structure to be
threaded by its binding partners (Mad1 or Cdc20).
The model suggested in this review represents the fi rst de-
scription of cell cycle regulation in which a partial unfolding of
the major signaling molecule and its refolding into an entirely
different conformation directs distinct downstream biochemical
outcomes. Future experiments designed to elucidate the unfold-
ing and refolding events that appear to determine the cycle of
Mad2 activation and silencing seem likely to provide important
insight into the elegant but complex mechanisms that faithfully
guard genome integrity at cell division.
We thank M. Lampson and J. Shah for their helpful comments on the manu-
script, L. Mayne for helpful discussion, and D. Slochower for his assistance
This work was supported in part by the National Institutes of Health
(research grants DP2OD002177 [J. Shorter], GM31847 [S.W. Englander],
and GM82989 [B.E. Black]), by a seed money research grant through the
Abramson Cancer Center (grant #IRG-78-002-30) from the American Cancer
Society (B.E. Black), and by a career award in the biomedical sciences from
the Burroughs Wellcome Fund (B.E. Black).
Submitted: 22 August 2008
Accepted: 29 October 2008
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