TRAF6 substrate profiling by Ub-BOD

Our method (Ub-POD) exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing biotin ligase BirA and an Avi tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potentially new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions.