Core MCC of Arabidopsis thaliana : structural dissection provides new insights into Mad2, BubR1 and CDC20

The spindle assembly checkpoint (SAC) is a mechanism executed mainly by the mitotic checkpoint complex (MCC) to monitor the anchoring of mitotic spindle microtubules to ensure correct chromosome segregation. Although knowledge of protein‒protein interactions in the plant cell cycle has progressed in recent years, information on the SAC mechanism in this context remains scarce. Here, we used bigBac, chemical crosslinking, mass spectrometry and structural analysis to identify protein‒protein interactions (PPIs) among AtCDC20, AtBubR1 (BMF2) and AtMad2, which are components of the core MCC26 in A. thaliana . Fifty-three nonredundant crosslinks were identified, with multiple binding sites in different domains and subdomains of these proteins, such as the β-propeller (AtCDC20), tetratricopeptide domain, and KEN boxes, including a new KET box (AtBubR1) and Horma domain (AtMad2). We also identified a hierarchy of interactions between these three proteins, with the potential to regulate signalling through the SAC molecular mechanism. Interactions that characterize the Mad2 tetramer, with the coupling of two dimers, were also identified. No PPIs were identified for AtBub1 (BMF1) or AtBub3:1, revealing a significant difference in MCC formation compared with that in other eukaryotes. Our findings provide a structural basis for core MCC formation in A. thaliana and offer new insights into the proteins CDC20, BubR1 and Mad2 in the context of the SAC.