Structural and functional characterization of the mad2a-kcash2 complex

MAD2A and KCASH2 are proteins involved in regulating cell cycle progression. MAD2A is a key component of the spindle assembly checkpoint (SAC), crucial for ensuring proper microtubule–kinetochore attachment and maintaining chromosomal stability during mitosis. Its expression is frequently dysregulated in tumors characterized by chromosomal instability. Our recent findings identify MAD2A as a novel interactor of the tumor suppressor KCASH2, a known inhibitor of the Hedgehog pathway. Preliminary in vitro co-immunoprecipitation assays show that KCASH2 binds MAD2A through its C-terminal region, promoting MAD2A ubiquitination and proteasomal degradation, thereby reducing its cellular levels. To further investigate this interaction, we employed a fragment-based modeling strategy focused on the putative MAD2A-binding region of KCASH2 and used AlphaFold to predict their complex structure. The resulting models revealed a conserved binding mode across different KCASH2 fragments, consistent with known MAD2A interactors. Based on these predictions, we reconstructed the full-length complex and assessed its structural stability through molecular dynamics (MD) simulations in GROMACS under both physiological and elevated temperature conditions. Interface analysis identified key residues likely mediating the interaction, guiding the rational design of mutants for future functional validation.