Fig. 1: Truncated NF-κB essential modulator (NEMO) is cleaved by 3CLpro in enzymatic assays.

a NEMO includes the α-helical domain 1 (Hlx1), the coiled-coil domain 1 (CC1), the α-helical domain 2 (Hlx2), the coiled-coil domain (CC2), a leucine zipper (LZ) domain, and the C-terminal zinc-finger (ZF). Human NEMO (hNEMO) truncated at site 215 and 247 was used in enzymatic assays with 3CLpro. A recognition site of cleavage is found at Gln231. b Cleavage of hNEMO_215–247 at 0.053 μg/μL (~13 μM) by 3CLpro at two concentrations. Reactions were incubated at 25 °C and aliquots were quenched at different times for analysis. The extent of proteolysis was quantified by LC-MS/MS. Apparent % cleavage was calculated by dividing the product peak area by the sum of the substrate and product peak areas. Error bars represent the range of duplicate enzymatic reactions. Statistics have been derived for n = 2 biologically independent experiments. c Multiple sequence alignment of peptide sequences of SARS-CoV-2 polyprotein and hNEMO. P1 site glutamine residues are shown in red. The peptide (P6 to P4’) used in the crystal structure is indicated beneath the sequences. d Coiled-coil pitch per residue computed with TWISTER²⁴ for NEMO in the PDB structures 6MI3 (region I)³⁴, 3CL3 (region II)³³, and 6YEK (region III)⁷³ is shown. Dashed lines indicate the regions I-III corresponding to these structures. The cleavage sites Gln83, Gln205, Gln 231, Gln304, and Gln313 are indicated with red arrows. The region corresponding to hNEMO_226–235, used in our X-ray structure determination is pointed out (violet arrow). e PAIRCOIL²⁵ prediction of coiled-coil propensity per residue for human and mouse NEMO (mNEMO). Lower P-scores implies greater likelihood of coiled-coil. Potential disordered binding regions predicted by ANCHOR²⁶ are shown in brown. The cleavage sites are depicted as in d. The region corresponding to hNEMO_226–235 used in our X-ray structure is depicted as in d.