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. 2017 Jan 17;18(3):611-623.
doi: 10.1016/j.celrep.2016.11.013. Epub 2016 Dec 13.

The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2

Paolo Swuec  1 , Ludovic Renault  1 , Aaron Borg  2 , Fenil Shah  3 , Vincent J Murphy  3 , Sylvie van Twest  3 , Ambrosius P Snijders  2 , Andrew J Deans  3 , Alessandro Costa  4
Affiliations

The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2

Paolo Swuec et al. Cell Rep. .

Abstract

Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.

Keywords: DNA repair; Fanconi anemia; cryo-electron microscopy; interstrand crosslink; mono-ubiquitination; ubiquitin ligase.

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Figures

None
Graphical abstract
Figure 1
Figure 1
FANCB-FANCL-FAAP100 under the Electron Microscope (A) Coomassie-stained SDS-PAGE gel of the purified BL100 complex. (B and C) Negative-stain (B) and cryo-EM (C) of the purified BL100 complex. (D and E) Representative negative-stain (D) and cryo-EM (E) two-dimensional class averages. Box size is 458 Å × 458 Å. (F) Top: BL100 forms an elongated, apparently 2-fold symmetric particle containing a dimerization core (blue) and two globular poles (red). Bottom: a cartoon representation of the assembly with overall particle dimensions. See also Figure S1.
Figure 2
Figure 2
Subunit Architecture of the FANCB-FANCL-FAAP100 Assembly Studied by Single-Particle Analysis (A) Silver-stained SDS-PAGE gel of the assembly containing an MBP N-terminally fused to FANCL. (B) Comparison between wild-type and MBP fused BL100 (negative-stain two-dimensional classes). Two MBP density features are marked with an orange arrowhead. Box size is 458 Å × 458 Å. (C) Cartoon representation of the BL100 complex with MBP in orange. (D) Coomassie-stained SDS-PAGE gel of the purified B100 subcomplex. (E) Negative-stain two-dimensional classes of B100. Box size is 458 Å × 458 Å. (F) Cartoon model of B100. In the absence of FANCL, the dimerization core remains unperturbed. The poles in the particle are more flexible. (G) Coomassie-stained SDS-PAGE gel of the isolated FAAP100 subunit. (H) Comparison between FAAP100 two-dimensional classes and two-dimensional classes of B100, focused on one pole in the particle. Box size is 458 Å × 458 Å. (I) Cartoon of the BL100 architecture derived from single-particle EM combined with subunit dropout experiments. See also Figures S2 and S3.
Figure 3
Figure 3
Subunit Architecture of the FANCB-FANCL-FAAP100 Assembly Studied by XL/MS (A) Inter-subunit crosslinks between the six-protein BL100 complex. The lengths of the subunits correspond to the lengths of the colored rectangles that they represent: blue, FANCB; green, FAAP100; orange, FANCL. Red lines represent intra-molecular crosslinks. Blue lines represent inter-molecular crosslinks. (B) Crystal structure of the human FANCL central domain with detected lysine residues highlighted in red (PDB: 3ZQS). All detected crosslinks in the FANCL region covered by the crystal structure map involve lysines that are surface exposed and are spaced within a range compatible with the spacer arm in the BS3 crosslinking agent. (C) Cartoon representation integrating the crosslinking/mass spectrometry and single-particle analysis characterization of the BL100 architecture. The total number of detected inter-\intra-molecular crosslinks is indicated for each cartoon. See also Table S1.
Figure 4
Figure 4
The FANCC-FANCE-FANCF Complex and Its Interaction with FANCB-FANCL-FAAP100 (A) Coomassie-stained SDS-PAGE gel of the CEF complex, containing an MBP N-terminally fused to FANCC. (B) Negative-stain two-dimensional classes of the of the MBP-CEF complex. Box size is 441 Å × 441 Å. (C) Coomassie-stained SDS-PAGE gel of the reconstituted and purified MBP-CEF/BL100 assembly. (D) Negative-stain two-dimensional classes of the reconstituted and purified (MBP)CEF-BL100 complex. Purple arrowheads point at the MBP-CEF density. Due to the flexible nature of the BL100-(MBP)CEF interaction, the MBP-CEF complex appears slightly smaller when BL100 is incorporated. A view of the isolated BL100 is shown, framed in white, for comparison. Box size is 580 Å × 580 Å. (E) Cartoon representation of the BL100-CEF architecture. See also Figure S4.
Figure 5
Figure 5
CEF Co-expression Stabilizes the FANCI-FANCD2 Interface (A) Coomassie-stained SDS-PAGE gel of the ID2 complex, with trace amounts of CEF complex. (B) Representative negative-stain micrograph of the ID2 assembly. Scale bar, 100 nm. (C) Two-dimensional class averages of the CEF-stabilized ID2 assembly. Box size is 441 Å × 441 Å. (D) CEF has a structural role in reshaping the ID2 interface, summarized in a cartoon. See also Figures S5 and S6.
Figure 6
Figure 6
Three-Dimensional Reconstruction of the CEF-Co-expressed FANCI-FANCD2 Assembly (A) Representative two-dimensional classes of the CEF-co-expressed ID2 complex matched to the two-dimensional projections of the electron density map generated from the ID2 crystallographic atomic coordinates (PDB: 3S4W). Box size is 350 Å × 350 Å. (B) Three orientations and a cut-through view of the X. laevis ID2 EM structure with docked mouse ID2 crystal structure. The fit is excellent, in particular, at the dimerization interface. Lysines targeted for mono-ubiquitination are highlighted in red (residue numbering refers to the mouse proteins, and the corresponding lysines in the X. laevis ortholog are indicated in parentheses). See also Figure S7.
Figure 7
Figure 7
Molecular Model for the Mechanism of FANCI-FANCD2 Mono-ubiquitination by the FA Core Complex
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