Fig. 8: A model for how tripartite assembly of pBub1:Cdc20:pMad1 in conjunction with Mad1^CTD fold-over orchestrates MCC formation.

a Model of pBub1:Cdc20:pMad1:C-Mad2:O-Mad2 complex (MCC-assembly scaffold) posed for MCC formation (Supplementary Movie 1). The model (Supplementary Data 1) was generated using PDBs 1GO4, 2V64, 6TLJ, 7B1F^21,23,26,90 and the AlphaFold2 models of Bub1^448–534:Mad1^CTD:Cdc20 and the folded model of Mad1^485–718, and displayed using ChimeraX. Residues for the Mad1 pThr716 and Cdc20^Box1, Cdc20^α1 Asp9 and Mad1 Lys619, Mad1 R617 and Bub1 pThr461 interactions are depicted as sticks, as well as the Bub1 ABBA interaction with the Cdc20 WD40 domain. b–d A schematic of catalytic MCC formation. b The doubly phosphorylated Bub1 CD1 domain targets the Mad1:C-Mad2 complex to kinetochores which then acts as a platform for O-Mad2 binding and O-to-C Mad2 conversion. Cdc20, on its own, likely exists in an autoinhibited state, which would impair the Cdc20:C-Mad2 interaction and MCC formation. c Phosphorylation of the C-terminus of Mad1 at Thr716 promotes its interaction with the N-terminus of Cdc20. Interaction between the WD40 domain of Cdc20 and the ABBA/KEN1 motif of Bub1 also occurs and this likely promotes Cdc20 kinetochore targeting and positions Cdc20 close to Mad1:C-Mad2. The Cdc20:pMad1^CTD interaction together with Mad1^CTD fold-over then promotes Cdc20-MIM accessibility and leads to Cdc20:C-Mad2 formation. d The Cdc20:C-Mad2 complex rapidly binds BubR1:Bub3 to form the MCC.