Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex

Abstract

The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA⁺ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

Keywords: SHLD2–SHLD3–REV7 complex; SHLD2–SHLD3–REV7–TRIP13 complex; SHLD3–REV7 complex; Shieldin assembly; TRIP13-mediated disassembly of Shieldin.

Fig. 1.

Crystal structure of SHLD3s–REV7 monomer complex reveals safety-belt topology and site-S interface. (A) Schematic drawing of human REV7 and SHLD3s fusion protein. The safety-belt segment of REV7 spans residues 152 to 180. (B) Purification of the SHLD3 (35 to 74)–REV7 monomer complex on an S200 gel filtration column. The major peak exhibited a mol. wt. = 32.5 kDa by SEC-MALS. (C and D) Two views of the overall structure of the SHLD3s–REV7 monomer complex. The N- and C-terminal halves of REV7 monomer are colored in light blue and green, respectively, while SHLD3s is colored in magenta. A black box highlights the site-S region in D. (E and F) Hydrophobic (E) and hydrogen bonding (F) interactions involving site-S. As shown in E, the bulky side chain of SHLD3 Phe-38 wedges into the hydrophobic pocket lined by REV7 residues Leu-186, Pro-188, Thr-191, and Tyr-202, while SHLD3 Trp-41 and Phe-42 stack tightly with the side chains of Glu-81, Glu-101, Thr-103, Lys-198, and Gln-200 of REV7. As shown in F, the backbones of SHLD3 Ile-39, Trp-41, Phe-42, and Pro-43 further interacts with the side chains of REV7 residues Glu-101, Lys-182, and Leu-173, and Ala-174 by hydrogen bonding.

Fig. 2.

Crystal structure of SHLD2.3–REV7₄ complex. (A) Schematic drawing of human REV7 and SHLD2.3 fusion protein. (B) Q column purification of the complex SHLD2.3 bound to REV7 yields two peaks labeled Q1 and Q2. (C) Size exclusion S200 purification of peak labeled Q1 and measurement of a 66.7-kDa molar mass for the major peak by SEC-MALS. (D) Size exclusion S200 purification of peak labeled Q2 and measurement of a 116.9 kDa molar mass by SEC-MALS. (E and F) Two views of the overall structure of SHLD2.3–REV7₄ complex. C-REV7, light blue; O-REV7, green; SHLD3, magenta; SHLD2, yellow. (G and H) Expanded views of the boxed segment in E (see G) and F (see H) of the complex highlighting the dimeric interface whereby SHLD2 β1–SHLD3 β1–REV7 β6 segments form a pair of β-sheets.

Fig. 3.

Comparison of closed C-REV7 and open O-REV7 conformations in the SHLD2.3–REV7₄ complex and intermolecular contacts. (A and B) Closed C-REV7 (A) and open O-REV7 (B) conformations in the SHLD2.3–REV7₄ complex. (C and D) Dimeric interface between C-REV7 and O-REV7 in the SHLD2.3–REV7₄ complex is mediated by multiple intermolecular hydrophobic (C) and hydrogen bonding (D) interactions. Trp-32, Leu-128, Val-132, and Ala-135 of O-REV7 build up a hydrophobic core with Leu-128, Vla-136, Pro-188, and Lys-190 of C-REV7 (C). Both Arg-124 of O-REV7 and C-REV7 form hydrogen bonds with Ala-135 of C-REV7 and Glu-35 of O-REV7, respectively, highlighting the importance of Arg-124 that is shown in previous reports. Lys-44, Ser-131, and Ala-135 of O-REV7 form additional hydrogen bonds with Asp-134, Lys-129, and Ser-36, respectively (D). (E and F) SHLD2–SHLD3 alignment in the O-REV7 is mediated by multiple intermolecular hydrophobic (E) and hydrogen bonding (F) interactions. Thr-147, Leu-149, and His-151 of O-REV7 and Val-5, Ile-6, Leu-7 His-8, and Leu-19 of SHLD3 build a hydrophobic core with Val-7, His-8, Ile-8, Phe-10, and Trp-11 of SHLD2 (E). The β-sheet is further stabilized by multiple backbone hydrogen bonds, while the backbone carbonyl oxygen of SHLD2 Phe-10 is recognized by the side chains of REV7 Tyr-63 and Thr-147 by hydrogen bonding (F).

Fig. 4.

Cryo-EM structure of SHLD2.3–REV7₄–TRIP13(E253Q) complex. (A) Schematic drawing of REV7, SHLD2.3, and TRIP13 (E253Q) proteins involved in complex formation. (B) Copurification of the complex formed by TRIP13(E253Q) hexamer and SHLD2.3–REV7₄ in the presence of ATPγS by size exclusion chromatography. (C) SDS-PAGE analysis of fractions from size exclusion chromatography. (D and E) The overall structure of the SHLD2.3–REV7₄–TRIP13(E253Q) complex with bound ATPγS shown in electron density (D) and ribbon (E) representations. The six subunits of TRIP13 are labeled A to F. (F and G) Views showing the insertion of the N terminus of C-REV7 into the central pore of the hexameric TRIP13 scaffold in electron density (F) and stick (G) representations. G highlights the interactions between the inserted N terminus (Asp8 to Val14) of C-REV7 and residues from color-coded subunits A to F of TRIP13.

Fig. 5.

Interactions between REV7 and TRIP13 in the structure of the SHLD2.3–REV7₄–TRIP13(E253Q) complex. (A and B) Interaction between the inserted N terminus (Asp8 to Val14) of C-REV7 and pairs of regions containing pore loops (213 to 241 and 255 to 289) from subunits A, B, C, D, and E of TRIP13 aligned in a spiral orientation in the complex. A shows a side view while B shows a top-down view of the inserted N terminus of C-REV7. (C) Interaction between the C-REV7 safety belt and the poly-E loop of TRIP13 in the complex. The main contacts occur between the monomer B poly-E loop and C-REV7 safety belt. The monomer A poly-E loop region is unstructured. (D) ATP activity assays of interfacial mutants of TRIP13 and SHLD2L.3–REV7. Data represent three independent experiments with mean ± SD.

Fig. 6.

Cryo-EM structure of SHLD2L.3–REV7₂–TRIP13(E253Q) complex. (A) Schematic drawing of REV7, SHLD2L.3, and TRIP13 (E253Q) proteins involved in complex formation. Note that SHLDL2.3 contains a longer version (residues 1 to 50) of SHLD2 compared with SHLD2.3 (residues 1 to 19 of SHLD2). (B) Copurification of the complex formed by TRIP13(E253Q) hexamer and SHLD2L.3–REV7₂ in the presence of ATPγS by size exclusion chromatography. (C and D) The overall structure of the SHLD2L.3–REV7₂–TRIP13(E253Q) complex with bound ATPγS shown in electron density representations. Three different classes of structures of the complex with different twist and tilt between the SHLD2L.3–REV7₂ and TRIP13 (E253Q) components are shown with side (C) and top (D) views.

Fig. 7.

Comparison of structures of SHLD2.3–REV7₄–TRIP13 and MAD2–p31^comet–CDC20–TRIP13 complexes. (A) Positioning of SHLD2.3-mediated O-REV7–C-REV7 dimer on the surface of TRIP13 following insertion of REV7^NT into the central pore of TRIP13 in the SHLD2.3–REV7₄–TRIP13 structure. (B) Positioning of MAD2–p31comet–CDC20 on the surface of TRIP13 following insertion of MAD2^NT into the central pore of TRIP13. (C) Electrostatic surface representation of the TRIP13 (surface potential at ±5 kT e⁻¹). Intermolecular contact patch between REV7 dimer and TRIP13 are highlighted by labeled yellow circles while the intermolecular contact patch between p31comet and TRIP13 is highlighted by a white circle. Monomers D and E use the consistent acidic surface for p31comet and O-REV7 binding, respectively. The TRIP13 pore is also indicated. The (D) REV7^NT and (E) MAD2^NT residues inserted into the TRIP13 pore.

Fig. 8.

Model of SHLD2.3–REV7 dimer complex remodeling mediated by the ATP-driven translocation of the TRIP13 hexamer. (A and B)Schematic of the proposed remodeling mechanism of TRIP13-mediated SHLD2.3–REV7 dimer. The fingers of TRIP13 grip the REV7^NT threaded segment tightly and the translocation of TRIP13 monomers draws the thread from REV7 into the channel in stepwise manner. (Cand D) Models of the SHLD2.3–REV7₄–TRIP13 complexes in basal state 0 and basal state 1 (before and after the first catalytic cycle, see more details in Movie S1). For clarity, only one copy of SHLD2.3–REV7 dimer is shown in a sphere representation. In basal state 0 (C), TRIP13 monomers A₀, B₀, and C₀ hold the C-REV7^NT, while monomer E₀ contacts O-REV7. As shown in D, the first cycle of ATP hydrolysis occurs in monomer E₀, which transforms from a compact ATP-bound state to the flexible apo-state E₁; the neighboring seam monomer F₀ binds one ATP molecule to adopt the ATP-bound F₁ state. These structural changes cause F₁ to climb to the top of the AAA⁺ spiral to push an anticlockwise rotation of the SHLD2.3–REV7 dimer, which renders O-REV7 to form new contacts with monomer D₁.