Determinants of conformational dimerization of Mad2 and its inhibition by p31comet - PubMed (original) (raw)
Determinants of conformational dimerization of Mad2 and its inhibition by p31comet
Marina Mapelli et al. EMBO J. 2006.
Abstract
The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.
Figures
Figure 1
Design of mutants impairing the O:C-Mad2 dimerization. (A) The ‘Mad2-template' model. O-Mad2 (red square) binds C-Mad2 (yellow circle) tightly associated with Mad1. This causes kinetochore recruitment of O-Mad2 (De Antoni et al, 2005a). O-Mad2 is quickly released and binds Cdc20. The model implies conformational dimerization of O-Mad2 and C-Mad2. (B) Sequence alignment of HORMA domains with secondary structure of HsMad2 in O-Mad2 and C-Mad2. We aligned 31 sequences of Mad2, Rev7 and Hop1 orthologues (Supplementary Figure 1). Mad2 conservation was evaluated on a larger alignment of 14 Mad2 sequences. Surface residues conserved in Mad2—but not in Rev7 and Hop1—were selected for mutation (blue boxes). (C) C-Mad2 bound to the Mad2-binding site of Mad1 (green, PDB ID 1GO4). The ‘safety belt' is colored red. Parts of the structure that are invariant in O-Mad2 and C-Mad2 are colored yellow. Side chains of mutated residues are dark blue. Dashed lines mark residues whose side chain is hidden from the current view. (D) Same representation as above for O-Mad2 (PDB ID 1DUJ). The first β-strand β1 (residues 1–15) is shown in light blue.
Figure 2
Visualization of the O-Mad2:C-Mad2 interaction. (A) Rationale of the solid-phase binding assay revealing the interaction of O-Mad2 with C-Mad2. (B) GST-Cdc20111−138 bound to GSH beads was incubated with Mad2wt, Mad2ΔC or both sequentially. Beads were washed and bound proteins analyzed by SDS–PAGE and Coomassie staining. Mad2ΔC cannot close on Cdc20111−138, while Mad2wt closes on the Cdc20 peptides and recruits Mad2ΔC onto the beads. The gel is a representative example of four independent experiments.
Figure 3
Mapping of residues involved in O-Mad2:C-Mad2 dimerization. (A) GST pull-downs (see Figure 2). Mad2wt and mutants were incubated for 1 h at 25°C with 1 μM GST-Cdc20111−138 preadsorbed on GSH beads to form C-Mad2. Excess Mad2 was washed away and equal amounts of O-Mad2ΔC were added. After 1 h, beads were washed twice and boiled in sample buffer. Bound species were separated by SDS–PAGE and Coomassie-stained. Red arrows indicate C-Mad2 mutants impaired in Mad2ΔC binding. (B) As in (A), but using CFP-Mad2ΔC instead of Mad2ΔC to obtain better separation from Mad2wt. (C) C-Mad2wt, C-Mad2F141A or C-Mad2R184A prebound to GST-Cdc20 were incubated with decreasing concentrations of O-Mad2ΔC. Binding of O-Mad2ΔC to Mad2wt was detected at concentrations as low as 0.5 μM. No binding of O-Mad2ΔC to C-Mad2F141A or C-Mad2R184A was observed at concentrations below 3 μM. (D) Equal amounts of Mad2wt were incubated with GST-Cdc20111−138 on GSH beads to create C-Mad2. O-Mad2ΔC and mutant variants were then added. (E) As in (D), but using CFP-Mad2wt as C-Mad2. The O-Mad2ΔC−T140E mutant deficient in binding to C-Mad2 is indicated with a red arrow. (F) C-Mad2wt prebound to GST-Cdc20 was incubated with decreasing concentrations of O-Mad2ΔC or O-Mad2ΔC−T140E. Binding of O-Mad2ΔC to C-Mad2wt was detected at concentrations as low as 0.5 μM. No binding of Mad2ΔC−T140E to C-Mad2wt was observed at concentrations below 5 μM. All gels are representative examples of at least three independent experiments.
Figure 4
p31comet is a competitive inhibitor of O-Mad2:C-Mad2 assembly. (A) Mad2ΔC and p31comet compete for C-Mad2wt bound to GST-Cdc20. Mad2ΔC (3 μM) and p31comet (0.3–3 μM) can separately bind to ∼ 1 μM Cdc20:C-Mad2wt on beads (lanes 2 and 6, respectively). Relative stoichiometries of Mad2ΔC and p31comet are indicated. When mixed together (lane 5), Mad2ΔC and p31comet are unable to enter a single complex. Already at equimolar concentrations, p31comet prevents O-Mad2ΔC from binding C-Mad2 (lane 5). (B) GST-Cdc20 pull-down assay with double C-Mad2 point mutants additionally harboring the R133A mutation. Experiments were repeated three times with identical results.
Figure 5
mad2T133A, mad2F134A and mad2K179A do not complement the deletion of the MAD2 gene in S. cerevisiae. (A) Numbering of equivalent human (Hs) and yeast (Sc) Mad2 residues discussed in the text. (B, C) Strains with the indicated genotypes were grown to log phase, arrested in G1 by α factor and released in fresh medium containing nocodazole. At the indicated times, cell samples were withdrawn for (B) FACS analysis of DNA contents, and (C) to score the percentage of budded and rebudded cells, as well as the percentage of sister chromatid separation.
Figure 6
NMR analysis of O-Mad2:C-Mad2 dimer. (A) Ribbon model of O-Mad2. Secondary structure is color-coded as in Figure 1. O-Mad2 residues required to bind C-Mad2 are in blue and ball-and-sticks. (B) C-Mad2 analyzed as in (A). (C) NMR chemical shift perturbation. The spectrum of O-Mad2ΔC (red) was superimposed with that of 1H,15N-labeled O-Mad2ΔC bound to equimolar unlabeled C-Mad2wt:Cdc20 (cyan). (D) The spectrum of C-Mad2wt (orange) was superimposed with that of 1H,15N-labeled C-Mad2wt bound to equimolar unlabeled O-Mad2ΔC (dark blue). (E) Summary of chemical shift perturbations monitored in 2D 1H,15N-HSQC experiments reported exclusively for residues that could be unambiguously assigned in the free and bound state. (F) The Cα atoms of residues in O-Mad2 whose amide's chemical shifts had changed are displayed as cyan spheres. (G) As in panel (F) for C-Mad2, with chemical shift perturbations indicated by dark blue spheres.
Figure 7
Model of O-Mad2:C-Mad2 interaction. (A) A cartoon summarizing the possible role of Mad1-bound C-Mad2 in the conversion of O-Mad2 into Cdc20-bound C-Mad2. Only the critical secondary structure elements are displayed. Panel 1 shows that the α3-helix of O-Mad2 docks on the α3-helix and β8 strand of C-Mad2. While the α3-helix remains docked onto C-Mad2, a displacement of the O-Mad2 core allows the passage of β1. The displacement is made possible by the long and flexible α3–β5 loop. After displacing β1, the C-terminal tail can move into its final position upon capturing Cdc20. (B) Mad2ΔN15, Mad2ΔC and Mad2ΔN15 were tested for association with C-Mad2wt prebound to GST-Cdc20111−138. Mad2ΔC binds readily to C-Mad2, while Mad2ΔN15 is a slow binder. The experiment was repeated at least three times with basically identical results.
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