Figure 3. The small molecule iNUB inhibits binding of NEMO_UBAN to linUb₂.

(a) DELFIA assay detection of StrepTagII-NEMO_UBAN (242–350) binding to His-tagged linUb₂ with or without iNUB. Increasing iNUB concentrations disrupted binding of NEMO to linUb₂ binding with an EC₅₀ of 9.26 μM. (b) NMR analysis of the NEMO_UBAN-linUb₂ interaction. ¹H, ¹⁵N HSQC spectra of 100 μM ¹⁵N-labeled linUb₂ in absence (orange) and in presence of unlabeled NEMO_UBAN C347S (258–350) (blue) at 1:1 stoichiometry. (c) Backbone amide resonances of Ub₂ resonances showing strongest changes upon NEMO_UBAN addition are annotated and indicated as spheres on the NEMO crystal structure (PDB 2ZVO). Red spheres indicate residues with unambiguous chemical shift assignments, while orange color shows amides, which could not be unambiguously assigned to the proximal or distal Ub moieties or showing signal overlap. Gray spheres are the amides of the corresponding residues in the other Ub module. (d) ¹H, ¹⁵N HSQC spectra of 100 μM ¹⁵N-labeled Ub₂ bound to the NEMO_UBAN (1:1) in the presence of DMSO (blue) or 290 μM iNUB (red). NMR signals of linUb₂ residues that showed reduced signal intensity upon binding to NEMO_UBAN are partially restored upon treatment with iNUB. (e) Mapping of Ub residues that show strongest effects upon iNUB addition onto the NEMO_UBAN structure as in (d). (f) Ratio of amide peak volume upon iNUB versus DMSO addition for the ¹⁵N-labeled Ub₂ complexed with unlabeled NEMO (1:1 stoichiometry).