Although the FANCI K522 and FANCD2 K559 side chains are embedded in the I-D interface (), the absence of I-D contacts next to each results in two tunnels, one for each lysine, that allow free access to bulk solvent from either side of the trough wall. In the monomeric proteins, both lysine side chains are fully solvent exposed.
The solvent-accessible tunnels of both sites are wide enough (~7 to 9 Å) and short enough to accommodate the four amino acid ubiquitin tail, whose C-terminus would be covalently linked to the lysine epsilon amino group (; fig. S7
). The ubiquitin tail is conformationally flexible (22
), and this should aid in the tail navigating the tunnels, while the ubiquitin structural domain is positioned outside the interface. It is also possible, however, that ubiquitination induces a conformational change or a rearrangement of the complex that fully exposes the mono-ubiquitination sites.
Mono-ubiquitination and phosphorylation sites of the ID complex
In principle, the tunnels can accommodate a ubiquitin residing either at the trough interior or exterior. The tunnel entrance at the trough interior (“top” entrance looking down the plane of ) is ~13 Å away from the lysine epsilon amino group. The entrance at the “bottom” is farther away at ~17 Å, although the relatively more open, funnel shape of this entrance could also accommodate part of the ubiquitin structural domain (; fig. S7 and SOM text
Irrespective of where the ubiquitin is positioned, the dimensions of the tunnels and entrances suggest that the ubiquitin tail and structural domains may contact both their conjugated protein and its hetero-dimerization partner. As these interactions would be repeated in a reciprocal fashion, the structure raises the possibility that ubiquitination may contribute to the stability of the ID complex. In this respect, we note that the mouse ID complex has a short half-life, as it dissociates during gel filtration chromatography at concentrations of ≤1 μM (fig. S8
Although the tunnels can readily accommodate the ubiquitin tail, they are too small for the active site of the ubiquitin-conjugating (E2) enzyme to access the lysine side chains. The structure thus suggests that ubiquitination either occurs on a cellular pool of monomeric FANCI and FANCD2, or it involves a process that opens up the I-D interface.
The sequestration of the lysine-ubiquitin isopeptide bond at the I-D interface would also protect it against de-ubiquitination by the FA-associated USP1 ubiquitin protease (23
). Coupled to a possible role of ubiquitination stabilizing I-D association, this may explain why cells lacking either FANCI or FANCD2 exhibit loss or reduction of the ubiquitinated form of the paralog (9