The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.
Bottom Line: The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown.We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro.However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.
Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.Show MeSH
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Mentions: We next wanted to understand the molecular determinants of the interaction between FANCL and ubiquitin. E2s bind ubiquitin non-covalently via a backside interaction that involves residues from the loop connecting strands β2 and β3 (30) (Fig. 2A). The dissociation constant between ubiquitin and the ELF domain suggests that crystallization of the complex would prove challenging. Indeed, despite extensive efforts, we were unable to obtain high-resolution diffracting crystals. Therefore, to understand the mode of ubiquitin binding by the ELF domain and whether it is similar to that seen in E2s, we set out to map the interacting surfaces using Nuclear Magnetic Resonance (NMR) spectroscopy. For both structural studies and ITC, milligram quantities of high-quality protein are required. The mammalian and vertebrate homologues of FANCL are not amenable to large scale soluble expression (20, 23); therefore, we used the more soluble invertebrate ELF domain from Drosophila, which shares ∼65% sequence similarity (19% identity) with the human ELF domain (19). First, we determined the solution structure of the ELF domain. Two-dimensional 15N-1H HSQC NMR of the 15N-labeled ELF domain yielded clear and resolved spectra, with excellent chemical shift dispersion, characteristic of a folded globular domain. We unambiguously assigned 76 out of 104 ELF residues using triple-resonance backbone datasets (Fig. 2B). Once we had determined the positions of each residue of the ELF domain in the spectra, we then titrated in increasing amounts of ubiquitin and recorded changes in the two-dimensional 15N-1H HSQC. Upon addition of ubiquitin, resonances were broadened to the extent that they were no longer visible, indicating a specific but transient interaction between the proteins (Fig. 3A). We then performed the reciprocal experiments by titrating increasing wild type ELF domain into 15N-labeled ubiquitin, and identified the binding site on ubiquitin (Fig. 3B). The interaction surface on the ELF domain involves a surface comprising residues Leu-53, His-54, Leu-74, Leu-76, and Leu-81 (Fig. 4, A and B). The interaction surface on ubiquitin is the Leu8-Ile44-Val70 central hydrophobic patch commonly recognized by ubiquitin-binding proteins (Fig. 4, A and B) (32). These results reveal a novel interaction surface on the ELF domain. This surface is not a relic of the E2-like fold, as it does not coincide with the predicted surface upon overlaying the structures (Figs. 2A and 4, C and D). To assess the requirement for residues in the interaction surfaces, we sought to validate our structural insights. We mutated residues involved in the binding, and assayed the resulting proteins for interaction using ITC. The ELF domain point mutant L81R completely abolishes binding (Fig. 5A), as does the ubiquitin mutant I44A (Fig. 5B). To test whether the leucine to arginine mutation on the exposed solvent-accessible surface of the ELF domain disrupts the folding of the domain, we performed two-dimensional 15N-1H HSQC NMR of the 15N-labeled L81R-ELF domain. These experiments yielded clear and resolved spectra, with excellent chemical shift dispersion, characteristic of a folded globular domain, with small changes compared with the wild-type spectra, consistent with a well-folded, stable, mutant protein (Fig. 5C).
Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.