Because purified recombinant Mad2 consisted of multiple conformers, structural studies on this protein relied on conformation-specific mutants that favored a particular conformation. The first structure of Mad2 determined was the solution structure of Mad2ΔC
, a mutant of Mad2 with its C-terminal 10 residues deleted that exclusively adopted the monomeric conformation (referred to as open/N1-Mad2 or simply O-Mad2; ) (Luo et al., 2000
). Using nuclear magnetic resonance (NMR) spectroscopy, the same study also showed that binding of the Mad2-binding motif of Cdc20 triggered a large conformational change on Mad2 and disrupted its oligomerization. The structures of Mad2 bound to a peptide ligand called MBP1 and to Mad2-binding domain of Mad1 were subsequently determined (Luo et al., 2002
; Sironi et al., 2002
). The ligand-bound conformation of Mad2 (referred to as closed/N2-Mad2 or simply C-Mad2; ) is strikingly different from that of O-Mad2. In C-Mad2, the C-terminal β-hairpin and the flexible tail of O-Mad2 rearranges into a new β-hairpin that has a hydrogen bonding network completely different from that of the original one (see below). This new β-hairpin translocates from one side of the Mad2 protein to the other and displaces the original N-terminal β-strand, which forms an additional turn in the central helix. The ligands, such as MBP1, Mad1, or Cdc20, are topologically trapped by the C-terminal β-hairpin of Mad2 and the preceding loop reminiscent of how car passengers are restrained by seat belts. Ligand dissociation requires partial unfolding of Mad2 (the detachment of β8’/8’’ from β5) and is thus expected to be exceedingly slow.
A Schematic Model Explaining Why O-Mad2 Is an Autoinhibited Conformer
Because the Mad2 dimer was more active in APC/C inhibition, it likely had a conformation that was distinct from O-Mad2 and favored Cdc20 binding. The structural studies of the Mad2 dimer had proved to be difficult, however. Luckily, mutation of a conserved residue in the αC helix, R133, to alanine disrupted the dimerization of Mad2 (Sironi et al., 2001
). Strikingly, Mad2R133A
had two monomeric conformers at equilibrium that were separable on an anion exchange column (Luo et al., 2004
). Based on NMR spectroscopy, the conformer that eluted in the low-salt fractions was O-Mad2. Structure determination of the conformer in the high-salt fractions revealed that it was unliganded C-Mad2, which resembled Mad1- or Cdc20-bound C-Mad2 but had an empty ligand-binding site (Luo et al., 2004
) (). The unliganded C-Mad2R133A
was more active in APC/C inhibition as compared to O-Mad2R133A
. Therefore, Mad2R133A
had two topologically and functionally distinct monomeric native folds at equilibrium under physiological conditions.
After extensive efforts, two recent studies finally revealed the structures of the Mad2 dimer (Mapelli et al., 2007
; Yang et al., 2008
). Yang et al. showed that Mad2 formed both a symmetric dimer that consisted of two unliganded C-Mad2 monomers and an asymmetric dimer that consisted of an O-Mad2 monomer and an unliganded C-Mad2 monomer (Yang et al., 2008
) (). This conformational heterogeneity explained the difficulty of previous structural analyses. The L13A mutant of Mad2 mainly formed the symmetric C-Mad2–C-Mad2 dimer. Structure determination of the Mad2L13A
dimer revealed a symmetric dimer interface that centered around the C-terminal end of the αC helix (). With the shortening a flexible loop in O-Mad2 and the addition of MBP1, Mapelli et al. managed to crystallize and determine the structure of the asymmetric O-Mad2–C-Mad2 dimer (Mapelli et al., 2007
) (). Collectively, these two structures explained why O-Mad2 could not form an O-Mad2–O-Mad2 symmetric dimer and provided the basis for the ability of Mad2 to form two types of dimers. Yang et al. also showed that Mad2L13A
was more active than the wild-type Mad2 in APC/C inhibition in vitro and in eliciting mitotic arrest in cells (Yang et al., 2008
). Therefore, unliganded C-Mad2, either as a monomer (as in the case of Mad2R133A
) or a dimer, is the functionally more active conformer of Mad2. We emphasize that, despite their obvious structural similarities, ligand-bound C-Mad2 and unliganded C-Mad2 differ in significant ways. Though both can form asymmetric dimers with O-Mad2, only unliganded C-Mad2 can form symmetric dimers.
The cartoon diagram in summarizes the equilibria and interconversions of the multiple conformers of Mad2 in vitro. During protein folding, the unfolded Mad2 folds into either O-Mad2 or unliganded C-Mad2. At the physiological temperature (37°C), O-Mad2 spontaneously converts to C-Mad2 with slow kinetics (t1/2
= 9 hrs) (Luo et al., 2004
), indicating that O-Mad2 is the high-energy state and that a large energy barrier separates the two states. In the case of Mad2R133A
, the molar ratio of O-Mad2:C-Mad2 at equilibrium is about 1:10, i.e. the ΔG of this reaction is about 1.4 kcal/mol (Luo et al., 2004
). In the case of the wild-type Mad2, O-Mad2 and C-Mad2 readily associate to form an asymmetric O–C dimer. C-Mad2 also forms a symmetric C–C dimer. The C–C dimer is less stable than the O–C dimer; the molar ratio of the two at equilibrium is about 1:3 (Yang et al., 2008
). The conversion rates between the C–C and O–C dimers are slow with a half-life of tens of minutes (our unpublished results), indicating that this structural transition also needs to overcome a high-energy barrier.
The Equilibria and Transitions of Mad2 Conformers
Both the C-Mad2 monomer and the C–C Mad2 dimer are more active in Cdc20 binding and APC/C inhibition as compared to the O-Mad2 monomer and the O–C Mad2 dimer. Why is C-Mad2 more active in Cdc20 binding and APC/C inhibition? Binding of either O-Mad2 or C-Mad2 to Cdc20 produces the same C-Mad2–Cdc20 complex, which has the same dissociation kinetics (off-rate). Therefore, the functional differences between O-Mad2 and C-Mad2 must be caused by differences in the association kinetics (on-rate). In O-Mad2, a vital structural element for Cdc20 binding, strand β6, is blocked by the C-terminal region of Mad2 (). It is not accessible for Cdc20 binding. O-Mad2 is thus an autoinhibited conformation. By contrast, β6 is exposed in C-Mad2 and is available for forming edge-on interactions with the Mad2-binding motif of Cdc20. The β8’/8’’ hairpin (the “seat belt”) can then detach from β5, wrap around Cdc20, and reattach to β5, trapping Cdc20 topologically. Thus, we propose that unliganded C-Mad2 binds to Cdc20 with a faster on-rate and a higher affinity as compared to O-Mad2.
In the O–C asymmetric Mad2 dimer, the β8’/8’’ hairpin is a major structural element of the dimer interface (). O-Mad2 thus impedes the movement of this hairpin in C-Mad2 and prevents it from encircling Cdc20, explaining the dominant-negative effects of O-Mad2 over C-Mad2 in the in vitro assays. In the C–C symmetric Mad2 dimer, the β8’/8’’ hairpin is not directly involved in dimerization and can complete the event of Cdc20 binding ().
Because O-Mad2 is a kinetically trapped high-energy state, binding of O-Mad2 to Cdc20 is not a simple equilibrium, as outlined in the following equation: O-Mad2 + Cdc20 <==> C-Mad2–Cdc20. Dissociation of C-Mad2–Cdc20 will mainly produce Cdc20 and unliganded C-Mad2, which is the thermodynamically stable state. Mathematical modeling and quantitative analysis of the spindle checkpoint need to take into account the O-Mad2 <==> C-Mad2 equilibrium when O-Mad2 binding to Cdc20 is considered. The correct binding reaction between Mad2 and Cdc20 at equilibrium is: O-Mad2 + Cdc20 <==> C-Mad2–Cdc20 <==> C-Mad2 + Cdc20.