Towards improving proximity labeling by the biotin ligase BirA

[Display omitted] •Screened for substitutions in the biotin binding site that modulate BirA activity.•An Arg-to-Lys substitution at position 118 increases biotinoyl-5′-AMP release.•Used methods to discriminate between self- and trans-biotinylation by BirA.•PCNA was identified as a Lamin A-proximal protein using BirA (Arg118Lys). The discovery and validation of protein–protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5′-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5′-AMP.