Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

Wenbin Ji; Yibo Luo; Ejaz Ahmad; Song-Tao Liu

A novel interface in MAD2 is employed for its association with MAD1-NTD or -CTD. A MAD2 molecule has two well-characterized interfaces for protein-protein interactions: the "safety belt" characteristic of C-MAD2 conformation to which MAD1-MIM and CDC20 binds (11, (38) (39) (40) , and the dimerization domain (primarily αC helix) that allows MAD2 to form O:C or C:C dimers or interact with p31 comet or BUBR1 (13, 32, 36, 41, 42) . MAD1-NTD and CTD bind to both MAD2 C10 and MAD2 L13A , suggesting no discrimination against either MAD2 conformation (Fig. 2b) . To further support this, wild type (WT) MAD2 existing as a mixture of both O and C conformers bind to all three MAD1 fragments, while another MAD2 mutant MAD2 S195D , predominantly in O conformation (43) , binds to MAD1-NTD or CTD but not MIM (Fig. 3a) . On the other hand, MAD2 LARQ (L13A/R133E/Q134A), a dimerization defective C-MAD2 mutant, associates with MAD1-NTD or CTD at similar levels as MAD2 L13A (Fig. 3b ). MAD2 N10 , which might mimic an intermediate conformation during O to C-MAD2 conversion (10) , binds to all three MAD1 fragments (Fig. 3c) . The above results suggest that the safety belt (or C-conformation), the dimerization domain (at least the RQ mutant) or the N-terminal 10 amino acids are not essential for MAD2 to interact with MAD1-NTD or CTD. Therefore a novel interface on MAD2, possibly shared by both O-and Cconformers, is employed for interactions with MAD1-NTD and CTD.