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. 2024 Sep 3;15:7692. doi: 10.1038/s41467-024-52076-0

Fig. 6. The YDDΦxΦ motif in NF-κB signaling partners of IKKs.

Fig. 6

a Motif peptide sequences from human IκBα, IκBβ, p100 and IRF7 proteins used in the pulldown experiments shown in (b). b Pulldown analyzes of the interactions between recombinant 6xHis-IKKα(10-667) EE (left panel) or 6xHis-IKKβ(1-669) EE (right panel) proteins and MBP-peptide fusions. IKKα and IKKβ were detected by Western blot (anti-His antibody) and MBP-peptides by Coomassie staining. These results were reproduced in at least two independent pulldown experiments. See also legend of Fig. 1b. c (Left panel) Domain architecture of p100. Disordered N-terminal region containing the YDDΦxΦ motif; RHD: Rel homology domain (residues 38-343) containing IKKα phosphorylation sites; ARD: ankyrin repeat domain (residues 487-758); DD: death domain (residues 764-851); disordered C-terminal region containing IKKα phosphorylation sites. (Right panel) Sequence alignment of residues 7-30 of human p100 showing conservation of the YDDΦxΦ motif. d Representative ITC binding isotherm for the IKKα/p100 pep interaction. The sequence of the synthetic p100 pep peptide is reported at the top. The data were analyzed by global fitting (see Table 1). The interaction was measured in two independent ITC titrations. Error bars correspond to the RMSD of the fitted curve and experimental values. See also legend of Fig. 2. e (Left panel) Representative dataset for the GPCA analysis of the interactions between full-length wt G1-IKKα and G2-p100 proteins. G2-p100 FL YD/SS: G2-p100 FL Y15S/D17S. (Right panel) Expression levels of G1-IKKα and G2-p100 constructs in HEK293T cells. See also legend of Fig. 1d. f (Left panel) Domain architecture of BANK1. TIR: Toll/interleukin-1 receptor/resistance protein domain (residues 25-153); DBB: Dof/BCAP/BANK domain (residues 200-327); ARD: ankyrin repeat domain (residues 342-408); disordered C-terminal region containing the YDDΦxΦ motif. (Right panel) Sequence alignment of residues 475-500 of human BANK showing conservation of the YDDΦxΦ motif in mammalian orthologs. g (Left panel) Representative dataset for the GPCA analysis of the interactions between full-length wt G1-IKKα and G2-BANK1 proteins. G2-BANK1 FL YD/SS: G2-BANK1 FL Y484S/D486S. (Right panel) Expression levels of G1-IKKα and G2-BANK1 constructs in HEK293T cells. Ctr.: non transfected cells. See also legend of Fig. 1d. h Sequence Logo showing the position-specific frequency of amino acids composing the motif in orthologues of IκBα, IκBβ, p100, IRF7 and BANK1. The Logo is based on the PSSM created using the binomial log10 scoring scheme from PSSMSearch (https://slim.icr.ac.uk/pssmsearch/) for the five validated peptides. Source data for this figure are provided as a Source Data file.