Molecular Mechanisms of Mitotic Checkpoint Complex Assembly onto Kinetochores

Abstract

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC), activates the mitotic checkpoint complex (MCC), to inhibit the E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Inhibition of the APC/C then delays premature chromosome segregation by preventing APC/C-mediated degradation of two key cell cycle regulators, cyclin B and securin. The MCC is composed of BubR1, Cdc20 and Mad2, and while their assembly is an intrinsically very slow process, in cells it is catalytically activated. Recent work points towards hierarchical recruitments of SAC proteins onto the outer kinetochore by means of a Mps1-dependent phosphorylation cascade, which creates a catalytic platform for MCC assembly.

This thesis investigates several mechanisms of catalytic MCC assembly in humans using a combination of biochemical assays and structural biology. Chapter 3 uses X-ray crystallography and NMR spectroscopy to explore the structure and function of the Bub1- Mad1 complex, including how sequential phosphorylation of the Bub1 CD1 domain by Cdk1 and Mps1 promotes kinetochore targeting of the Mad1:C-Mad2 complex. Chapter 4 investigates how Mad1 C-terminal phosphorylation by Mps1 promotes juxtaposition of SAC proteins for MCC assembly. This includes using NMR to gain detailed structural insights into how phosphorylation of Mad1 promotes its interaction with both the N-terminus of Cdc20, as well as a region within Bub1 just C-terminal to its CD1 domain. Chapter 5 investigates the structure of the Mad1:C-Mad2:O-Mad2 complex by cryo-EM and reveals a mechanism of Mad1CTD fold-over which has import implications for MCC assembly. Chapter 6 sets the premise for ongoing work on the molecular mechanisms of Mad2 conversion from the open to closed state by NMR.