Figure 4. iNUB binds and alters the conformation of the NEMO_UBAN.

(a) Microscale Thermophoresis (MST) assays to determine binding of iNUB to NEMO_UBAN or linUb₂. iNUB binds to Myc-NEMO_UBAN-ZF-StrepTagII (242–350) with a K_D of 2.14 μM. (b) ¹H,¹⁵N HSQC spectra of 50 μM ¹⁵N-labeled linUb₂ in the absence (black) and presence of 1 mM iNUB (red). (c) NMR analysis of the NEMO_UBAN-iNUB interaction. ¹H,¹⁵N TROSY NMR spectrum of 117 μM ¹⁵N-labeled NEMO_UBAN (258–350) with DMSO (black) or 870 μM iNUB (red). Amide signals that are affected upon iNUB addition are indicated. Asterisks indicate unassigned NMR signals that are affected by iNUB. (d) Mapping of residues affected upon iNUB addition onto the NEMO_UBAN crystal structure (PDB 2ZVO).