Mapping the energy landscape of a fold‐switching protein <scp>MAD2</scp>

The mitotic arrest deficiency 2 (MAD2) exists in inactive and active forms under physiologic conditions. In its active conformation, MAD2 binds to the cell division cycle protein 20 (Cdc20) and prevents the separation of duplicated chromosomes. In the inactive conformation, the C-terminal region of MAD2 covers the binding motif for the Cdc20 target. Here, we investigated the MAD2 activation mechanism using structure-based models (SBMs) simulations, amino acid coevolution, and structural frustration analysis. MAD2 switches between active and inactive conformations while maintaining core stability. Simulations reveal an intermediate state during the transition consistent with recent time-resolved NMR experiments. Coevolution analysis captures native contacts for both states. These native contacts, present in both conformations, compete, driving transitions between different protein states. This competition leads to a frustrated energy landscape. Frustration analysis further shows that highly frustrated residues are present in both conformations, particularly in the fold-switching segments.