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)1,2. 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) conformer3,4,5. 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 p31comet, an SAC antagonist6, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2)7,8,9,10. Here, we present a mechanism that explains how TRIP13–p31comet disassembles the MCC. Cryo-electron microscopy structures of the TRIP13–p31comet–C-MAD2–CDC20 complex reveal that p31comet recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2NT) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2NT within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2NT to push upwards on, and simultaneously rotate, the globular domains of the p31comet–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 p31comet. 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.
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Acknowledgements
This work was funded by MRC and CR-UK grants to D.B. C.A. is an EMBO Advanced Fellow. We thank A. Boland for comments on the manuscript; S. Chen, C. Savva and G. McMullan for help with EM data collection; and J. Grimmett and T. Darling for computing.
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Nature thanks K. Corbett, H. Yu and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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C.A. cloned bacterially expressed p31comet, MAD2 and TRIP13 wild type and mutant constructs, purified proteins, performed the protein complex reconstitutions and biochemical analysis and mutagenesis. C.A. prepared EM grids, analysed EM data and determined the three dimensional reconstructions. C.A. collected EM data with the help of L.C. C.A. fitted coordinates and built models. D.B. directed the project. C.A. and D.B. wrote the manuscript with input from L.C.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical characterization of MAD2-containing complexes with TRIP13.
a, TRIP13 and p31comet can extract O-MAD2 from MCC and APC/C–MCC regardless of APC15. Western blot showing the disassembly reactions together with the respective input material (i) of APC/C-bound MCC (APC/C–MCC), in either the absence (lanes 1–2) or presence of APC15 (lanes 3–6) and MCC alone (lanes 7–10) (experimental design is shown on top). Negative control reactions (lanes 3, 4 and 9, 10) were performed with the TRIP13(E253Q) mutant. MAD2 levels were detected with an anti-MAD2 antibody. Loading controls of APC4–STREP, APC15, CDC20, TRIP13 and BUB3 were detected with antibodies specific for STREP, APC15, CDC20, TRIP13 and BUB3, respectively. BUBR1 (lanes 7–10) was detected with an anti-STREP antibody. b, Western blots showing eluted size exclusion (Superdex 200 10/300 column) fractions of the MCC and MAD2 remodelling reactions catalysed by TRIP13–p31comet in the context of free MCC and APC/C–MCC. The fractions corresponding to (i) APC/C–MCC, (ii) MCC, (iii) p31comet–C-MAD2 and (iv) monomeric C-MAD2 are shown. A reference gel for size exclusion column elution fractions corresponding to p31comet–C-MAD2–CDC20–MBP, p31comet–C-MAD2 and monomeric C-MAD2 is shown in Extended Data Fig. 2a. c, Analysis of TRIP13–p31comet complexes with the MCC using size exclusion chromatography in the presence of ATP. Coomassie-stained gel showing the gel filtration fractions (chromatogram above) of p31comet–TRIP13 complexes in complex with MCC (fraction 8 is the p31comet–TRIP13 complex with C-MAD2–CDC20 and fraction 9 is the BUBR1–BUB3 complex). Input material (i) is shown on the left. d, MCC binds p31comet and not TRIP13 alone. Coomassie-stained gel showing the gel filtration fractions of the MCC (top gel) and in the presence of p31comet (middle gel) and TRIP13(E253Q) (lower gel). e, Chromatogram (top) and SDS–PAGE (bottom) of the gel filtration performed with the TRIP13(E253Q)–p31comet–C-MAD2–CDC20 complex in the presence of ATPγS. Experiments in a–e were performed in triplicate with similar results. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 2 Biochemical assay for TRIP13–p31comet-catalysed O-MAD2 generation.
Shown are size exclusion (Superdex 200 10/300 column) chromatograms and corresponding Coomassie-stained gels for the Mad2 remodelling reaction catalysed by TRIP13–p31comet. a, Reference chromatograms and Coomassie-stained gels for (i) p31comet–C-MAD2–MBP–CDC20 (brown trace), (ii) p31comet–C-MAD2 (blue trace) and (iii) monomeric O-MAD2 (red trace). Chromatograms and gels are colour-coded. Monomeric O-MAD2 elutes in fractions 22–24, whereas p31comet–C-MAD2 elutes in fractions 17–19. b, Western blots for the products of the reaction of TRIP13 with (i) wild-type p31comet and MAD2 (orange trace) and (ii) wild-type p31comet and mutant C-MAD2 (C-MAD2Δ7 (seven N-terminal residues deleted)). c, Western blots for the products of the reaction with (i) mutant C-MAD2LEE, wild-type TRIP13 and p31comet (yellow trace), (ii) mutant TRIP13(E269R/D272R) and wild-type p31comet and C-MAD2 (green trace) and (iii) mutant p31comet α3–4 and wild-type TRIP13 and C-MAD2 (black trace). Experiments in a–c were performed in triplicate with similar results. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 3 Cryo-EM analysis and resolution of TRIP13 complexes in this study.
a, Left, gallery of 2D class averages of TRIP13–p31–substrate showing different views representative of 50 2D classes. Right, a typical cryo-EM micrograph of TRIP13–p31–substrate representative of 3,630 micrographs. b, Fourier shell correlation (FSC) curves are shown for all the cryo-EM reconstructions determined in this study. c, Local resolution maps calculated with RESMAP41 of the TRIP13–p31–substrate complex. d, Cryo-EM density of the TRIP13–p31–substrate reconstruction shown as in Fig. 1b. e, g, Representative density quality for the ATPγS (e) and β-strand (g). In e, critical residues for the TRIP13 catalytic site are indicated: WA (Walker A), WB (Walker B), S1 (sensor 1), S2 (sensor 2) and RF (Arg finger). f, Close up of the TRIP13 pore loops interacting with C-MAD2NT from the cryo-EM density of TRIP13–p31–substrate. EM density for C-MAD2 shown in black mesh, TRIP13 in transparent coloured surface.
Extended Data Fig. 4 3D classification of TRIP13–p31–substrate full data set.
a, Local refinement (see Methods) by applying a mask covering TRIP13A/B/C/D/E monomers. b, c, 3D class averages obtained by classification (see Methods) of the TRIP13–p31–substrate full data set (b) and TRIP13-p31-substrate (c). The percentages relative to the total number of TRIP13-p31-substrate particles are shown.
Extended Data Fig. 5 Comparative analysis of TRIP13 structures.
a, Comparison of TRIP13 cryo-EM structure (right, this study) and previous TRIP13 crystal structures (left, C. elegans PCH217; middle, human TRIP1318). b, Comparison of TRIP13 monomers within the TRIP13 cryo-EM structure. RMSDs between TRIP13A and other TRIP13 monomers are indicated in the inset table. A superimposition of all six TRIP13 monomers is shown, colour-coded as in Fig. 1. TRIP13F differs from all the other monomers in the relative orientation of the small and large AAA+ domains. Its conformation relative to TRIP13A is shown at the lower right. The open conformation prevents nucleotide binding. (The sensor 2 residue (S2) is positioned too far from the ATP-binding site.) Lower left, a superimposition of TRIP13F onto an open subunit C (grey) of the PCH2 structure17. c, Conformational differences in pore loop-1 between the PCH2–ADP17 complex (grey) and the cryo-EM TRIP13–ATPγS complex (orange) (this study).
Extended Data Fig. 6 Structures of MAD2.
Structural context of MAD2. a, C-MAD223; b, in the TRIP13–p31comet–C-MAD2 complex (this work); c, O-MAD223; d, the Schizosaccharomyces pombe mitotic checkpoint complex42 composed of C-MAD2, CDC20 and BUBR1/MAD3 (BUB3 is not shown); e, p31comet–C-MAD2 complex19. In all figures the regions of MAD2 that reposition during the O-MAD2 to C-MAD2 transition are coloured blue and red for N-terminal (residues 1–16) and C-terminal (158–204) regions, respectively. In O-MAD2 these are the N terminus (MAD2NT) and β1 strand (blue) and C-terminal β7–β8 hairpin (red). In C-MAD2 these are MAD2NT including the αN helix and first turn of αA (blue), and the C-terminal β8′–β8′′ hairpin, safety belt and C terminus (MAD2CT) (red). On conversion of O-MAD2 to C-MAD2, the β1 strand is displaced and replaced by the β8′–β8′′ hairpin. Residues 13–15 of β1 form an additional turn at the N terminus of αA in C-MAD2. The C-MAD2 ligand is coloured purple. MBM, MAD2-binding motif; MBP1, high-affinity MAD2-binding peptide43,44.
Extended Data Fig. 7 TRIP13 interacts with p31comet through a composite interface formed of monomers D and E.
a, Details of the interaction between TRIP13 and p31comet. Left, schematic of TRIP13–p31comet interactions. Right, structure showing details of the main electrostatic contacts between TRIP13 and p31comet. Above, schematic of the domain architecture of TRIP13 and p31comet. A row of aspartates on α7 engages the conserved safety belt motif residues Arg233 and Arg237 of p31comet. The adjacent Lys162 contacts a Glu-rich loop (111–121) in TRIP13 that is disordered in previous TRIP13 crystal structures17,18. In our structure the Glu-rich loop lies directly above pore loop-1. Glu104 and Asp105 of the TRIP13NTD-ATPase domain linker, immediately preceding the Glu-rich loop, contact Arg227 and Lys229 of the p31comet safety-belt, agreeing with the importance of Lys229 for TRIP13-p31comet interactions in vitro17 and in vivo20. On monomer E, the same acidic patch of α7 of the large AAA+ domain contacts basic residues at the N terminus of α3 of p31comet. b, Details of the interaction of the p31comet α3–4 loop with TRIP13 subunit E. Seven basic residues shown were deleted and the mutant p31comet(α3–4 loop) was tested in MAD2 remodelling assays and for assembly of a TRIP13–p31comet–C-MAD2 complex. c, Multiple sequence alignment of the p31comet α3–4 loop. d, Deletion of the nine N-terminal residues of MAD2 (MAD2(Δ9)), and mutation of the p31comet α3–4 loop (p31comet(α3–4 loop)) do not disrupt TRIP13–p31comet–C-MAD2 complex assembly. Coomassie-stained gel showing the gel filtration fraction of wild-type and relevant mutant TRIP13–p31comet–C-MAD2 complexes purified by size exclusion chromatography. Experiment in d was performed in triplicate with similar results. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 8 Conservation of TRIP13 pore loops and MAD2NT.
a–c, Multiple sequence alignment of TRIP13 pore loop-1 (a) and pore loop-2 (b) and the N-terminal region of MAD2 (c).
Extended Data Fig. 9 Models of the TRIP13–p31comet–C-MAD2 complexes in basal state 0 and basal state 1 (before and after the first catalytic cycle).
a, Basal state 0 (similar to Fig. 4a). Superscripts on TRIP13 subunit labels denote basal state. b, Showing the conformational change of the TRIP13 hexamer after one catalytic cycle (basal state 1) superimposed on the p31comet–C-MAD2 substrate before catalysis (as in basal state 0). The upward movement of the E and F subunits clashes with p31comet and C-MAD2. The pore loop residues of E and F subunits shift by 30 Å and 13 Å, respectively. c, Close up view of b showing the clash between the F1 and E1 subunits of TRIP13 in basal state 1 with C-MAD2 and p31comet, respectively (as in basal state 0). d, Basal state 1 with TRIP13 and p31comet–C-MAD2 in the remodelled conformation (as in Fig. 4b) and now with the whole of the p31comet–C-MAD2–CDC20 substrate repositioned onto the new C1 and D1 interface and the αA helix unwound by one turn. e, As in d but rotated by 60° to show the p31comet–C-MAD2–CDC20 substrate in the same view as in a. f–j, Panels showing TRIP13 and only C-MAD2NT, connection to the C-MAD2 αA helix and proposed unwinding of the C-MAD2 αA helix. f, MAD2NT and αA in basal state 0 with view of TRIP13 (in basal state 1 conformation) as in e. g, TRIP13 in basal state 1 conformation as in e with the whole p31comet–C-MAD2 substrate repositioned onto the C1–D1 interface. Note that the αA helix of C-MAD2 has not been unwound and C-MAD2NT (residues 2–12) is shifted along with the globular domain of C-MAD2 (C-MAD2GD). h, Basal state 1 with C-MAD2NT in the basal state 0 position (that is, unshifted relative to basal state 0) and αA of C-MAD2GD as p31comet–C-MAD2 is repositioned on the C1–D1 interface (but the αA helix is not unwound). This shows the 11 Å break between the Cα atoms of Thr12 of C-MAD2NT and Leu13 of C-MAD2GD. C-MAD2NT is shifted down the TRIP13 pore by two residues relative to g. i, Close up view of the C-MAD2NT and connection to the αA helix. The view is the same as in h and it superimposes the positions of C-MAD2NT as in basal state 1 and basal state 0, showing a 7.6 Å distance between the Cα atoms of Thr12 of C-MAD2NT in the two states. This indicates the extent of required unwinding of the αA helix. j, The first turn of the αA helix unwinds to reconnect Thr12 of C-MAD2NT with Leu13 of the C-MAD2 αA helix. Model building was guided by insights from cryo-EM structures of the AAA+ ATPases VAT45, Vps446 and Hsp10447 that indicate a processive hand-over-hand mechanism in which the AAA+ motor translocates along the axis of the substrate. ATP hydrolysis propagates a sequential conformational change within the hexameric ring that converts the conformation of each subunit to that of its clockwise neighbour viewed from p31comet.
Supplementary information
Supplementary Figure 1
Original source images for all data obtained by electrophoretic separation: Coomassie stained SDS PAGE and western blots
Video 1: Overview of the TRIP13 and the TRIP13:p31comet:C-MAD2:CDC20 complex.
This video shows an overall view of the apo TRIP13 and then the TRIP13:p31comet:C-MAD2:CDC20 complex (TRIP13-p31-substrate complex) (basal state/class 2).C- MAD2NT enters the central pore of TRIP13
Video 2: Molecular mechanism of C-MAD2 remodelling by TRIP13 in conjunction with p31comet.
Two catalytic cycles are shown. Each catalytic cycle results in the translocation of two residues of C-MAD2NT through the TRIP13 central pore in conjunction with unwinding of one turn at the N-terminus of the MAD2 αA helix. This breaks contacts to the β8’-β8” hairpin (Ser16 of αA to His191 of β8’-β8” is shown) to destabilize the C-MAD2 β-sheet, promoting the conversion of C-MAD2 to O-MAD2
Video 3: ATP hydrolysis by TRIP13 promotes a conformational change of the TRIP13 ring and a clash with p31comet:MAD2.
This video shows how ATP-dependent translocation of TRIP13 along Mad2NT causes subunits E and F to clash with p31comet and C-MAD2, respectively
Video 4: Comparison of basal and active states of the TRIP13:p31comet:C-MAD2:CDC20 complex.
This video shows conformational change between the basal and activated states of the TRIP13:p31comet:C-MAD2:CDC20 complex. The E and F monomers change conformation on transition from the basal to activated state, moving towards p31comet:C-MAD2
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Alfieri, C., Chang, L. & Barford, D. Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13. Nature 559, 274–278 (2018). https://doi.org/10.1038/s41586-018-0281-1
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DOI: https://doi.org/10.1038/s41586-018-0281-1
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