Fig. 7. NMR-monitor conversion of O-Mad2 to C-Mad2 shows that Cdc20^MIM greatly enhances the rate of Mad2 conversion.

a C-Mad2 (orange) bound to Cdc20 (green) from PDB 6TLJ⁹⁰, with Mad2 Trp167 and Trp75 side chains depicted as sticks. The middle panel shows a ¹H, ¹⁵N 2D HSQC of ¹⁵N-labelled O-Mad2 (blue), C-Mad2 (red), and Cdc20^MIM-bound C-Mad2 (orange). All Mad2 samples contain an R133A mutation to prevent Mad2 dimerisation²³. The C-Mad2 sample additionally contains an L13A mutation which favours the C-Mad2 state⁵⁷. Cdc20^MIM-bound C-Mad2 sample was prepared by adding a 1:2 excess of Cdc20^MIM peptide to O-Mad2 with an R133A mutation. The indole N-H resonances for Mad2 tryptophan side chains were used as reporters for Mad2 conformation and are boxed in the spectra. The right panel shows the close-up views of the two tryptophan indole N-H resonances of O-Mad2 (blue), empty C-Mad2 (red), and Cdc20^MIM-bound C-Mad2 (orange). As Cdc20^MIM is entrapped by the safety-belt, the changes in the chemical environment of Trp167 and Trp75 result in distinct chemical shifts of their side chain resonances. b The MIM motif of Cdc20 induces the conversion of O-Mad2 into C-Mad2. Using the side chain resonances of Trp167 and Trp75 as reporters, the O-to-C conversion of Mad2 was traced in a 24-h time course at 25 °C. The schematics show the conformation of Mad2 as indicated by the tryptophan side chain resonances and the full spectra are shown in Supplementary Fig. 13a, b. O-Mad2 in the presence of Cdc20-MIM converts to Cdc20^MIM-bound C-Mad2 (orange) within 30 min, while O-Mad2 alone takes up to 24 h to fully convert to C-Mad2 (red).