Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination (2022)
Figure 5

Fig. 5. The extended interface of FANCI's ubiquitin with FANCD2 is required for enhanced protection against USP1-UAF1.

A. Interactions of FANCI and FANCD2 with the ubiquitin conjugated to the other ID2 subunit, in DNA-bound IUbD2, IUbD2Ub (PDB: 6VAE), ID2Ub (PDB: 6VAF) and IUbD2Ub (PDB: 6VAE) structures (Wang et al, 2020). Dotted straight lines indicate hydrogen bonding. Both FANCD2 and FANCI interact with ubiquitin's hydrophobic I44 patch (residues L8, I44, H68 and V70; all labelled in highlighted-yellow font) and additionally with residues F45 to G47 of ubiquitin. However, the ubiquitin conjugated to FANCI has a more extensive interface with FANCD2. This extended interface is formed by interactions of FANCD2 α10 - α13 helices (predominant interacting residues highlighted in boxes) with residues R74, T9 and K11 of ubiquitin (shown in black font). The ubiquitin-FANCD2 interface may be further extended via interactions between residues K33 and E34 of ubiquitin with K165 and R174 of FANCD2, as shown in IUbD2Ub-DNA structure. For direct comparison of corresponding interactions, the same orientation for all ubiquitins (both FANCI-conjugated and FANCD2-conjugated) was achieved by aligning: ID2Ub-DNA and IUbD2Ub-DNA structures to the ubiquitin of IUbD2-DNA structure, and subsequently, the IUbD2Ub-DNA structure to the ubiquitin of ID2Ub-DNA structure as well.

B. Clustal O multiple sequence alignment of human, mouse, rat, chicken, frog and zebrafish FANCD2 amino-acid sequences, focused on a region encompassing α11-13 helices of FANCD2 in human IUbD2-DNA structure (helical regions shown on top). Identical residues among various species are highlighted red, whereas residues in positions displaying 83% similarity/identity are shown in red font. Residues of FANCD2 interacting with FANCI's ubiquitin in both IUbD2-DNA and IUbD2Ub-DNA structures (extended FANCD2-ubiquitin interface) are indicated with blue circles.

C-D. Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown. D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.