Structural Analysis of Human FANCL, the E3 Ligase in the Fanconi Anemia Pathway

Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2011; 286(37):32628-37. DOI: 10.1074/jbc.M111.244632
Source: PubMed


The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

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    • "Although the interaction between UBE2T and FANCL E3 ligase was shown in a yeast two-hybrid screen (Machida et al., 2006), it has eluded detection in living mammalian cells. The UBE2T-FANCL dissociation constant was measured as 0.454 mM by isothermal titration calorimetry, but this required analysis at 8 C (Hodson et al., 2011). Furthermore , it was shown that cocrystallization of UBE2T and FANCL required their fusion (Hodson et al., 2014), consistent "
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    ABSTRACT: Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 11/2014; 9(5). DOI:10.1016/j.celrep.2014.10.058 · 8.36 Impact Factor
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    • "The existence of FA subcomplexes is consistent with earlier work (Garcia-Higuera et al., 2001, 2000; Ling et al., 2007; Medhurst et al., 2006; Thomashevski et al., 2004) and could represent different stoichiometries within the assembled complex, assembly intermediates, and/or functional modules. For example, although FANCL has been shown to directly bind FANCD2 and FANCI (Cole et al., 2010; Hodson et al., 2011; Seki et al., 2007), C-E-F could represent a functional module that regulates these substrate interactions via FANCE-FANCD2 associations or impinges upon substrate accessibility by USP1 (Gordon et al., 2005; Nookala et al., 2007; Pace et al., 2002; Polito et al., 2014). FANCF could act as a scaffold to anchor both the A-G module (Lé veillé et al., 2004) and the FANCM-FAAP24-FAAP10-FAAP16 module implicated in chromatin targeting, suppression of crossover recombination, and checkpoint signaling (Collis et al., 2008; Deans and West, 2009; Rosado et al., 2009). "
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    ABSTRACT: Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
    Molecular Cell 06/2014; 54(5):858-869. DOI:10.1016/j.molcel.2014.05.001 · 14.02 Impact Factor
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    • "Maximal FANCD2 ubiquitination shows a strong dependence on FANCL in our reconstituted system (Figure 1C). FANCL interacts transiently with FANCD2 and FANCI, and a hydrophobic patch in the middle portion of FANCL has been shown to be important for these protein interactions (22). Mutation of the W212 and L214 residues located in this hydrophobic patch attenuates the ability of FANCL to interact with FANCD2 and FANCI (22). "
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    ABSTRACT: FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku198 · 9.11 Impact Factor
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