Structural Insights into Linear Tri-ubiquitin Recognition by A20-Binding Inhibitor of NF-κB, ABIN-2

Recognition of linear polyubiquitin by specific ubiquitin-binding proteins plays an important role in mediating nuclear factor-κB (NF-κB) signaling. A20 binding proteins, ABINs, recognize linear polyubiquitin and A20 through UBAN and AHD1, respectively, for the inhibition of NF-κB activation. Here we report the crystal structure of the AHD1-UBAN fragment of ABIN2 in complex with linear tri-ubiquitin, which reveals a 2:1 stoichiometry of the complex. Structural analyses together with mutagenesis, pull-down, and isothermal titration calorimetry assays show that the hABIN2:tri-ubiquitin interaction is mainly through the primary ubiquitin-binding site, and also through the secondary ubiquitin-binding site under a high local protein concentration. Surprisingly, three ubiquitin units could form a right-handed helical trimer to bridge two ABIN2 dimers. The residues around the M1-linkage are crucial for ABIN2 to recognize tri-ubiquitin. The tri-ubiquitin bridging two ABIN2 dimers model suggests a possible higher-order signaling complex assembled between M1-linked polyubiquitinated proteins, ubiquitin-binding proteins, and effector signaling proteins in signal transduction. [Display omitted] •Crystal structure of hABIN2 in complex with linear tri-ubiquitin with a 2:1 ratio•hABIN2 preferentially uses the primary Ub-binding site to bind a tri-ubiquitin•A tri-ubiquitin molecule bridges two hABIN2 dimers at a high protein concentration•A helical arrangement of linear polyubiquitin allows a higher-order complex assembly Lin et al. report the first crystal structure of hABIN2 in complex with linear tri-ubiquitin, which shows the ubiquitins with M1-linkage could form a right-handed helical trimer when bridging two hABIN2 dimers, and thus allow the assembly of a higher-order signaling complex with A20.