Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13

Abstract

The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome). Unattached kinetochores control MCC assembly by catalysing a change in the topology of the β-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31, an SAC antagonist, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2). Here, we present a mechanism that explains how TRIP13-p31 disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31-C-MAD2-CDC20 complex reveal that p31 recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2 within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2 to push upwards on, and simultaneously rotate, the globular domains of the p31-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 β-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.

HORMA domain proteins and a Pch2-like ATPase regulate bacterial cGAS-like enzymes to mediate bacteriophage immunity

“…Monomer F is positioned at the "bottom" of the spiral, and as such shows the most significant conformational differences compared to the other five subunits. Overall, the structure of the Ec Pch2 EQ hexamer closely resembles that of other substrate-engaged AAA+ ATPases, including mammalian TRIP13 (Alfieri et al, 2018).…”

“…As noted above, eukaryotic TRIP13 recognizes its HORMA domain substrate Mad2 through the adapter protein p31 comet (Alfieri et al, 2018;Ye et al, 2017). Our structure of Ec CdnC:HORMA:Pch2 EQ reveals that in this complex, CdnC serves as the adapter, binding the top face of the Pch2 hexamer at the interface of monomers E and F (Fig.…”

“…A number of recent studies have reported moderate-resolution (3.2-5.0 Å) cryo-electron microscopy structures of AAA+ ATPase remodelers engaged with their substrates, including mammalian TRIP13 bound to a p31 comet :Mad2 complex (Alfieri et al, 2018;Deville et al, 2017;Gates et al, 2017;Han et al, 2017;Puchades et al, 2017;Ripstein et al, 2017;White et al, 2018;Yu et al, 2018). These structures have revealed that active AAA+ remodelers adopt a right-handed spiral or "lock-washer" conformation, with 4-5 nucleotide-bound subunits forming a tight helical spiral and engaging an extended substrate peptide with their "pore loops".…”

“…These structures have revealed that active AAA+ remodelers adopt a right-handed spiral or "lock-washer" conformation, with 4-5 nucleotide-bound subunits forming a tight helical spiral and engaging an extended substrate peptide with their "pore loops". In the case of TRIP13, the structures revealed that the "adapter" protein p31 comet binds the top surface of the TRIP13 hexamer, thereby positioning the substrate Mad2 such that its extended N-terminus drapes into the hexamer pore for engagement and remodeling (Alfieri et al, 2018;Ye et al, 2017). In our structure of Ec CdnC:HORMA:Pch2 EQ , the Ec Pch2 EQ hexamer adopts a right-handed spiral conformation, with four subunits (monomers A-D) bound to ATP, and two subunit (monomers E and F) unbound ( Fig.…”

“Amyloid‐beta accumulation cycle” as a prevention and/or therapy target for Alzheimer's disease

“…Congenital mutations causing cohesinopathy in humans result in conditions with intellectual disability, deformity, and proneness to cancers, such as Cornelia de Lange syndrome and Roberts syndrome (Cucco & Musio, ; Zhu & Wang, ). Other aneuploidogenic conditions include mosaic variegated aneuploidy syndrome, which is caused by a mutation in (a) a mitotic checkpoint component BubR1/Bub1B (type 1); (b) CEP57, which encodes a centrosomal protein (type 2) (Snape et al, ); or (c) TRIP13, which is involved in mitotic checkpoint complex silencing via Mad2 liberation (Alfieri, Chang, & Barford, ; Kaisari et al, ). A reduction in BubR1 protein in mice (BubR1 H/H mice) resulted in a systemic and neuronal progeria condition (Choi et al, ) and in impairment of adult hippocampal neurogenesis (Yang et al, ).…”