The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex

Abstract

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the ‘knitting model’ that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

Molecular mechanism of Mad2 conformational conversion promoted by the Mad2-interaction motif of Cdc20

“…SAC signalling is mediated by the MCC 2 , whose assembly is rate-limited by formation of C- Mad2:Cdc20. SAC catalysts 20,27,28 generate an MCC-assembly scaffold that functions to present the exposed Cdc20 MIM to its binding site on Mad2 27,28,30–32 . In contrast to the rapid association of Cdc20 MIM peptides to empty C-Mad2, full-length Cdc20 does not readily bind empty C-Mad2 28 , likely due to the steric hindrance of threading the long N-terminal segment of Cdc20 through the closed safety-belt of C-Mad2 28 .…”

“…Juxtaposing the accessible Cdc20 MIM adjacent to O-Mad2 by the MCC-assembly scaffold involves the dimerisation of an O-Mad2 molecule with Mad1-bound C-Mad2 28,[30][31][32] . C-Mad2 binds to Cdc20 MIM and the MIM of Mad1 (Mad1 MIM ) through comparable mechanisms [13][14][15][16] .…”

“…Unattached kinetochores activate the SAC signal by catalysing formation of the MCC 21 , in which the key catalysts of MCC-assembly are the protein kinase Mps1, the Mad1:C-Mad2 heterotetramer and Bub1 20,27,28 . These catalysts generate and contribute to an MCC-assembly scaffold that accelerates formation of the C-Mad2:Cdc20 complex [27][28][29][30][31][32] . Mps1 activity, stimulated by the Ndc80 complex at unattached kinetochores 33,34 , phosphorylates Knl1 and Bub1, thereby promoting Bub1 interactions with Mad1 and Cdc20.…”

“…Mps1 activity, stimulated by the Ndc80 complex at unattached kinetochores 33,34 , phosphorylates Knl1 and Bub1, thereby promoting Bub1 interactions with Mad1 and Cdc20. This scaffold both promotes the accessibility of Cdc20 MIM (through a proposed conformational change of Cdc20 28,30,35,36 ) and juxtaposes Cdc20 MIM adjacent to its cryptic binding site on an O-Mad2 molecule interacting with C-Mad2 of the Mad1:C-Mad2 complex 28,[30][31][32] (Fig. 1d).…”