Mad2-containing complexes. (A and B) The displacement of the C-terminal segments in C-Mad2 exposes a new β 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 β6 and newly formed β7′. 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 β8′ 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 interconversion 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 fit would increase the forward O-Mad2→C-Mad2 rate, whereas the conformational selection of C-Mad2 would reduce 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.

Comparing the Mad2 conformational switching reaction to models for prion propagation. (A and B) Models for prion propagation adapted from Aguzzi (2004). (C) A simplified reaction scheme for Mad2 conversion catalyzed by self–self interactions.

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 chromatids align at the metaphase plate early in mitosis and wait for ∼20 min before chromatid separation in anaphase. Upon final 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 sufficient 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.

Mad2 unfolding and refolding considerations. (A) Free energy reaction landscape for Mad2 interconversion through a partially folded intermediate 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 intermediate 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 difference 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.