Structure of an intermediate conformer of the spindle checkpoint protein Mad2

Abstract

The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.

Cdc20: At the Crossroads between Chromosome Segregation and Mitotic Exit

“…Similarly, the interaction of Cdc20 with Mad2 is critical for the SAC. Mad2 is a 23.5-kDa protein that contains a Hop1p, Rev7p, and MAD2 (HORMA) domain of multiple conformational states, including an open (o-Mad2) and a closed (c-Mad2) conformation [36,37]. In the latter conformational state, Mad2 binds to Cdc20 and to its upstream regulator Mad1.…”

Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

“…3b). MAD2 N10 , which might mimic an intermediate conformation during O to C-MAD2 conversion (10), binds to all three MAD1 fragments (Fig. 3c).…”

“…C-MAD2 then utilizes its dimerization domain to recruit O-MAD2 and converts the latter into C-MAD2, resulting in signal amplification for the mitotic checkpoint (6,7,12). The conversion may go through multiple intermediate states (I-MAD2) (6,7,10,13,32). At least two structures of I-MAD2:C-MAD2 dimers have been solved, one possibly representing the initial O-MAD2 "docking" complex and the other containing a later stage of I-MAD2 state (MAD2 N10 ) (10,32).…”

“…The solved MAD2 N10 structure indicates a topology similar to O-MAD2 but its internal core more closely resembles that of C-MAD2 (10). However, one long suspected intermediate state with partially unfolded N-or Ctermini, which is more amenable for MAD2 refolding or its interaction with CDC20, has not been identified based on studies of various MAD2 conformational dimers therefore was considered as only a fleeting state (10). Such a state, as suggested (29,30), may be coupled with formation of the CDC20:MAD2 complex or the MCC, making the MAD2 O-C conversion reaction more rapid and energy favorable.…”

“…During prometaphase intracellular C-MAD2 concentration is increased to promote formation of the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex/cyclosome (APC/C) (1)(2)(3)(4). In the current model, the MAD2 O-C conversion is catalyzed by a 2:2 MAD1:C-MAD2 tetramer localized at unattached kinetochores, whereby cytoplasmic O-MAD2 hetero-dimerizes with the C-MAD2 moiety in the catalyst and morphs into C-MAD2 through unknown mechanism but possibly involving some intermediate folding states (I-MAD2) (6,7,10).…”