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

Laurence P.C Lewis,Ambrose R Cole,Jennifer A Miles,Charlotte Hodson,Andrew Purkiss,Helen Walden

doi:10.1074/jbc.m111.244632

Laurence P.C Lewis, Ambrose R Cole + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.244632

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Abstract

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.

Highlights

Fanconi anemia (FA)4 is an X-linked or autosomal recessive disorder with a range of clinical presentations including bone marrow failure and high incidence of cancer [1]
The structure of the human central FANCL domain (URD) adopts the same overall topology as the Drosophila double RWD (DRWD) domain, confirming that the FANCL proteins belong to the UBC and RING superfamilies and are not WD40 proteins as predicted [17]
The human central domain does differ from the DmFANCL as it does not have a DRWD but contains a UBC fold fused to an RWD fold (URD)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Previous efforts to structurally characterize human FANCL had proved unsuccessful, but the understanding of the domain architecture of DmFANCL allowed us to design new constructs. Proteins were expressed in Escherichia coli BL21 cells (Invitrogen) in lysogeny broth medium supplemented with antibiotics and with 0.5 mM ZnCl2 in the case of the RING domain. Cells were harvested by centrifugation and lysed by sonication on ice (10-s bursts followed by 30 s on ice, repeated four times) in 100 mM Tris, pH 8.0, 500 mM NaCl, 20 mM imidazole, 250 ␮M tris(carboxyethyl)phosphine (10 ␮M ZnCl2 added when purifying the RING domain). Purified proteins were concentrated to 20 mg/ml (central domain), 8 mg/ml (RING), and 15 mg/ml (Ube2T), flash-frozen in liquid nitrogen, and stored at Ϫ80 °C. Crystallization and Structure Determination—Human FANCL central domain crystals were grown by hanging drop vapor diffusion at 4 °C, in space group C2, with cell dimensions a ϭ 147.6 Å, b ϭ 102.5 Å, c ϭ 65.8 Å, ␣ ϭ 90, ␤ ϭ 94.09, ␥ ϭ 90. Both a low-resolution pass and a high-resolution pass were collected (3.5 and 1.75 Å, respectively)

Data collection and refinement statistics

RESULTS

DISCUSSION