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. 2020 Jan 3;295(1):250-262.
doi: 10.1074/jbc.RA119.011464. Epub 2019 Dec 3.

Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair

Yaxin Dai  1 , Fan Zhang  2 , Longge Wang  1 , Shan Shan  3 , Zihua Gong  4 , Zheng Zhou  5
Affiliations

Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair

Yaxin Dai et al. J Biol Chem. .

Abstract

Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2-2.3 Å resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal α-helix (αC-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved αC-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the αC-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 αC-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 "safety belt" region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3-REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.

Keywords: DNA repair; REV7; crystal structure; isothermal titration calorimetry (ITC); mitotic arrest deficient 2 like 2 (MAD2L2); nonhomologous end-joining repair; protein–protein interaction; shieldin complex subunit 3 (SHLD3); structural biology; surface plasmon resonance (SPR).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
SHLD3 employs REV7-binding domain to interact with REV7. a, schematic domain architecture of SHLD3. The RBD and EIF4E-like domain of SHLD3 are depicted in magenta and green, respectively. The protein sequences of the presumptive REV7-binding motif 1 (RBM1) and RBD used in crystallization are displayed with the defined prolines in two RBMs colored in red. The secondary structure illustration of the RBD region is also shown above the protein sequence. b, isothermal titration calorimetric analysis of three different SHLD3 fragments binding to REV7. Raw data and fitting curves of the integrated data for various SHLD3 proteins are shown together with corresponding KD values. c, overall structure of REV7 in complex of SHLD3 presented in cartoon mode. The SHLD3 fragment is shown in magenta, and the REV7 molecule is colored light blue, except for the C-terminal region of REV7, which is termed the safety-belt region (residues 153–207), highlighted in pale green. The N-terminal loop and αC-helix region of SHLD3–RBD are indicated.
Figure 2.
Figure 2.
Structural basis of SHLD3(RBD)–REV7 complex. a, stereo view of the interaction details between SHLD3 and REV7. Colors of molecules correspond to those in Fig. 1c, and residues involved in the interaction are labeled. The hydrogen bonds are shown as yellow dotted line. b and c, Ni pulldown assays using His–REV7 co-expressed with SHLD3(29–83) fragments. After rounds of extensive washing, the bound proteins were resolved by SDS-PAGE stained with Coomassie Brilliant Blue (upper panel). His–REV7(R124A) was used as control sample. The bottom plot panel shows the relative abundance of the pulled down SHLD3 proteins. The relative band intensities of SHLD3(29–83) divided by band intensities of REV7 were normalized to those of the control. Values are represented as means ± S.D. from three independent experiments. One-tailed Student's tests are indicated: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001. d, representative ITC results of the interactions between SHLD3 RBM mutants and REV7. Representative binding curves for WT, P53A/P57A, P53A/P58A, and P57A/P58A SHLD3 are shown as black circle, yellow circle, green circle, and blue circle, respectively. The binding affinity of each mutant is shown accordingly. e, 293T cells were transfected with plasmids encoding Myc-tagged SHLD3 WT and its mutants together with the plasmid encoding SFB-tagged REV7. Immunoprecipitation reactions were conducted using S protein beads and then subjected to Western blotting using the indicated antibodies. f, beads coated with bacterially expressed MBP-fused SHLD3 was incubated with cell lysates containing exogenously expressed SFB-tagged REV7 WT and its mutants. Immunoblotting experiments were carried out using the indicated antibodies.
Figure 3.
Figure 3.
αC-helix region of SHLD3–RBD underpins REV7–SHLD3 interaction. a, structural comparison of SHLD3 with other REV7-binding peptides. All REV7-binding segments are displayed in cartoon mode, and REV7 molecules are represented in surface mode. The angles between helix and loop of individual peptides are indicated. Left to right: SHLD3 (this study); REV3–RBM1 (PDB code 3ABD); CAMP (PDB code 5XPT); and REV3–RBM2 (PDB code 6BC8). b, structural superimposition of four REV7-binding partners. The REV7 molecule is represented in surface mode, and four fragments are shown in cartoon mode, with colors corresponding to those in a. c, representative ITC results of the interactions between SHLD3 mutants and REV7. The binding affinity and corresponding fitting curves are presented. Corresponding thermodynamic parameters are shown in Fig. S4b. d, Ni pulldown assays using His–REV7 co-expressed with Ile-60 mutants of SHLD3. The procedures are the same as depicted in Fig. 2 and under “Experimental procedures.” The results of SDS-PAGE are shown in the left panel, and the right scatter plot shows the relative abundance of the pulled down SHLD3 proteins. Data are represented as means ± S.D. from three independent experiments. One-tailed Student's tests are indicated: **, p < 0.01; ***, p < 0.001. e, 293T cells were transfected with plasmids encoding SFB-tagged SHLD3 WT and its mutants. Immunoprecipitation reactions were conducted using S protein beads and then subjected to Western blotting using the indicated antibodies. f, Ni pulldown assays using His–REV7 mutants co-expressed with SHLD3(29–83) fragments. The procedures are the same as described above. The results of SDS-PAGE are shown in the left panel, and the right scatter plot shows the relative abundance of the pulled down SHLD3 proteins. Data are represented as means ± S.D. from three independent experiments. One-tailed Student's tests are indicated: ns, not significant; ***, p < 0.001.
Figure 4.
Figure 4.
Binding kinetics of SHLD3–REV7 complex. a–d, SPR sensograms obtained during and after injection of MBP–REV7 or corresponding mutants over the immobilized SHLD3 chip surface. The model-fitting curve is displayed as black lines that overlay to the original sensograms. The concentrations of the MBP–REV7 proteins are indicated. e, statistics of association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD) of each reaction calculated from sensograms.
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