Allosteric mechanism for site-specific ubiquitination of FANCD2

DNA damage repair is implemented by proteins that are coordinated by specialised molecular signals. One such signal in the Fanconi Anemia (FA) DNA-interstrand crosslink repair pathway is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multi-protein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate E2 Ube2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyses ubiquitination by rewiring Ube2T’s intra-residue network to influence the active site. Consequently, a basic triad unique to Ube2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of Ube2T can be engineered to enhance FANCL catalysed FANCD2-FANCI di-monoubiquitination without compromising site-specificity.