The AAA+ ATPase TRIP13 remodels HORMA domains through N‐terminal engagement and unfolding

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X‐ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31 comet . We show that p31 comet binding to the TRIP13 N‐terminal domain positions the disordered MAD2 N‐terminus for engagement by the TRIP13 “pore loops”, which then unfold MAD2 in the presence of ATP. N‐terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro , and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13‐mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13. Synopsis The AAA+ ATPase TRIP13 and the adaptor p31 comet catalyze conformational conversion of MAD2 to support both activation and inactivation of the spindle assembly checkpoint (SAC). Crosslinking mass spectrometry and crystallography show how TRIP13 engages MAD2 and releases its target peptide‐trapping “safety belt” during SAC silencing. A TRIP13 hexamer binds a single p31 comet :MAD2 substrate complex. TRIP13 pore loops engage the disordered MAD2 N‐terminus for unfolding. The MAD2 N‐terminus is required for TRIP13‐mediated conformational conversion in vitro , and for SAC function in cells. MAD2‐related meiotic HORMAD proteins are also TRIP13 substrates requiring disordered N‐termini for proper function in meiotic prophase. TRIP13 may function by disrupting interactions between the MAD2 N‐terminus and safety‐belt region that stabilize its closed conformation. Graphical Abstract Crosslinking mass spectrometry and crystallography reveal how TRIP13 and the adaptor p31 comet recognize MAD2 and release its “safety belt” during mitotic checkpoint silencing.