Insights into Mad2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric Mad2 Dimer

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Abstract
In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/CCdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint. Chromosome missegregation during mitosis results in the gain or loss of chromosomes in the next generation of cells and can contribute to birth defects or cancer. A cellular surveillance system called the spindle checkpoint ensures that accurate chromosome segregation occurs by inhibiting the activity of the anaphase-promoting complex or cyclosome (APC/C) until all sister chromatids have achieved proper attachment to the mitotic spindle. The spindle checkpoint protein Mad2 binds to Cdc20, an activator of APC/C, and inhibits the complex. The Mad2 protein can adopt either an open or closed conformation. The conformational switch in Mad2 is critical for Cdc20 binding and APC/C inhibition, and is regulated by the protein Mad1. We report the crystal structure of the symmetric Mad2 dimer, which is made up of two closed monomers, and is active in APC/C-Cdc20 inhibition. Mad1 seems to facilitate the open–closed conformational switch of Mad2, and we present a unified model to explain Mad1-assisted Mad2 activation in the spindle checkpoint.