Structure of the mitotic checkpoint complex

Abstract

In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.

APC/C is an essential regulator of centrosome clustering

“…Second, Eg5 carries several putative KEN 19 and D-box 20 motifs at the C-terminal. In particular, the KEN and D-box motifs at 1,022-1,024 and 1,047-1,050 (RLPL) amino acids, respectively, are in close proximity to promote cooperative binding 21 and, importantly, these motifs are highly conserved among different species (Fig. 2b).…”

“…APC/C ubiquitination assays with recombinant human APC/C and CDH1 were performed as previously described 21 . Recombinant APC/C was a kind gift from Jing Yang, Barford lab.…”

The Biochemistry of Mitosis

“…Cdc20 and Cdh1 are WD40 family members and the aminoterminal residues of the D-box bind between blades 1 and 7 of the WD40 domain; the carboxy-terminal residues bind to APC10 (He et al 2013). A second degron, Lys-Glu-Asn, called the KEN-box, binds to the top surface of the WD40 domain (Kraft et al 2005;Buschhorn et al 2011;Chao et al 2012). …”

Understanding the structural basis for controlling chromosome division

“…The Mad2 β8 -β8 hairpin (red) that forms the Mad2-Mad3 interface, repositions on conversion from O-Mad2 to C-Mad2. The N terminus of the WD40 domain is indicated [34].…”