Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1

• Published in: EMBO reports Vol. 22; no. 7; pp. e52242 - n/a
• Main Authors: Fischer, Elyse S, Yu, Conny W H, Bellini, Dom, McLaughlin, Stephen H, Orr, Christian M, Wagner, Armin, Freund, Stefan M V, Barford, David
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05-07-2021; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Anaphase; Anaphase-promoting complex; Assembly; Asymmetry; Bub1; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Chromosome Segregation; Coils; Crystal structure; Dipoles; Domains; EMBO06; EMBO40; Kinases; Kinetochores; Kinetochores - metabolism; Mad1; Mad2 Proteins - genetics; Mad2 Proteins - metabolism; Metaphase; Mitotic checkpoint complex; Peptides; Phosphorylation; Signal Transduction; Signaling; Spindle Apparatus - metabolism; Spindle assembly checkpoint; Stoichiometry; Mad1; Spindle assembly checkpoint; Bub1; Cell cycle; Mitotic checkpoint complex
• ISSN: 1469-221X; 1469-3178
• DOI: 10.15252/embr.202052242

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1
CTD
) bound to two phosphorylated Bub1
CD1
peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1
CD1
peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1
CTD
and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.
Synopsis
Activation of the mitotic checkpoint complex (MCC) is orchestrated by a sequential phosphorylation-dependent signalling cascade. This study reveals the molecular mechanism for kinetochore targeting of the Mad1-Mad2 complex.
The crystal structure of the C-terminal domain of Mad1 (Mad1
CTD
) bound to phosphorylated Bub1
CD1
peptides explains how phosphorylation creates a direct interaction with Mad1
CTD
.
Bub1
CD1
pThr461 not only directly interacts with Arg617 of the conserved RLK motif of Mad1
CTD
but also stabilizes the Bub1
CD1
helix dipole.
In solution only one Bub1
CD1
binds to the Mad1 homodimer.
Inherent asymmetry within Mad1
CTD
likely controls Bub1 binding and stoichiometry.
Graphical Abstract
Activation of the mitotic checkpoint complex (MCC) is orchestrated by a sequential phosphorylation-dependent signalling cascade. This study reveals the molecular mechanism for kinetochore targeting of the Mad1-Mad2 complex.