The AAA + ATP ase TRIP 13 remodels HORMA domains through N‐terminal engagement and unfolding

Abstract

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 We show that p31 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 , 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.

Mechanistic insight into TRIP13-catalyzed Mad2 structural transition and spindle checkpoint silencing

“…6 g). Therefore, consistent with the results of Corbett and coworkers 58 , the N-terminal region of Mad2 is required for TRIP13 binding and proper MCC disassembly. This result does not, however, indicate that the N-terminus of Mad2 directly engages the pore loop of TRIP13 or that the unfolding of Mad2 by TRIP13 starts at the N-terminus.…”

“…5 ). The fractionation profiles of TRIP13 WT in the presence of ATP were very similar to those reported previously 58 . Thus, in the presence of ATP, the G320A mutation does not appear to disrupt the oligomerization of TRIP13.…”

“…Our structure of human TRIP13 bound to ADP is virtually identical to those of human apo-TRIP13 or TRIP13 bound to ATP 58 (Supplementary Fig. 2b ).…”

“…Recently, Corbett and workers suggested that TRIP13 engages the N-terminal region of Mad2 through the pore loop and unfolds Mad2 from the N-terminus 58 . The most crucial evidence for Mad2 N-terminal unfolding by TRIP13 is the crosslinking data performed with the TRIP13 W221C mutant.…”

“…TRIP13 then induces the conformational change of C-Mad2 to O-Mad2 in a process that requires ATP hydrolysis 55 . The structures of both human TRIP13 and its Caenorhabditis elegans homolog PCH-2 have been determined 55 , 58 . Chemical crosslinking and functional studies further suggest that TRIP13 might promote the structural transition of Mad2 through engaging and unfolding the N-terminal region of Mad2 58 .…”