Extended Data Fig. 5: Charges within the phospho-loop destabilize the open conformation of D2-I.
From: The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination
(a) Surface representation of the M. musculus crystal structure of D2-IWT (PDB 3S4W, FANCD2 in blue, FANCI in magenta), in which the phospho-loop was modelled at the dimerization interface of the open D2-I complex (left). The electrostatic potential of the phospho-loop region (calculated in ChimeraX) is shown. The scale used to represent the electrostatic potential is a gradient from −10 (red) to +10 (blue) kT, and regions with a net charge of 0 are colored in white. The surface to which the three S/TQ motifs (ATR phosphorylation sites) map to is mostly neutral, whereas the proximal FANCD2 surface exhibits negative electrostatic potential (middle). The introduction of bulky, charged groups could cause steric or charge-based repulsions that disrupt FANCI interaction with the negatively-charged surface of FANCD2 at the interface. A zoomed-in view of this interface is shown on the right (cartoon representation) where selected residues (sticks) and their H-bonding network (dashed blue lines, generated in ChimeraX) are depicted. The three ATR target residues (yellow) in the phospho-loop (orange) along with other selected residues (tan) are shown as sticks. (b-d) Control reactions of the 160-minute time-point of the monoubiquitination assay shown in Fig. 5a-c performed in the absence of DNA (b), presence of 100 nM dsDNA (c), or presence of 5 µM 44-bp dsDNA (d). Two independently performed assays have been carried out and showed the same result. FA-CC, FA core complex.
