Fig. 4.

Substitution of lysine residues in the active site moderately increases biotin labeling.A, crystal structure of Aquifex aeolicus BirA (PDB ID: 2EAY; https://www.uniprot.org/uniprot/O66837) with residues R40G, K102, and K103 depicted. B, human embryonic kidney (HEK) cells were transfected with IFNLR1-BioID2 (Δ63, L41S, and L46F) with additional mutations at K36, K44, and K102. At 48 h post-transfection, cells were labeled for 1 h with biotin at the indicated concentrations and probed as described previously. C, densitometry of biotin labeling via streptavidin–HRP normalized to HA expression. B and C, n = 3 experiments. ∗p < 0.05, ∗∗p < 0.01 versus IFNLR1-Δ63 (L41S, L46F) by ANOVA. HA, hemagglutinin; HRP, horseradish peroxidase; IFNLR1, interferon-lambda receptor 1; PDB, Protein Data Bank.