Structure of Human Mad1 C‐terminal Domain Reveals Its Kinetochore‐Targeting Function

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. The kinetochore‐bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood, however. To understand the functional importance of the C‐terminal region of Mad1, we solved the crystal structure of human Mad1 C‐terminal domain (CTD) and found that it formed a homodimer and had a fold similar to that of the known kinetochore‐targeting domain of Spc25 (a subunit of the Ndc80 complex). Indeed, Mad1‐ ∆CTD was defective in kinetochore targeting in human cells. The mutagenesis studies validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results thus reveal novel functions of Mad1‐CTD in kinetochore targeting. Deletion of CTD does not abolish Mad1 kinetochore localization, however. Non‐overlapping Mad1 fragments retain detectable kinetochore targeting. Therefore, our results indicate that CTD is a part of an extensive kinetochore‐binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.