Towards improving proximity labeling by the biotin ligase BirA

Abstract

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.

Keywords: BioID: biotinylation; BirA; Nuclear lamina; Protein–protein interactions; Proximity ligation.

Fig. 1.

Proximity ligation protein labeling. Overview of enzyme-based proximity labeling. An enzyme capable of generating a reactive intermediate is expressed as a fusion with a protein of interest (yellow) that results in appropriate subcellular targeting. Label addition results in proximal labeling of proteins bound directly (red) and indirectly (green) to the protein of interest. Proteins are subsequently solubilized, and the proximity-modified proteins are enriched on beads by virtue of the label. The proteins labeled in this manner are identified by mass spectrometry. (B) Crystal structure of BirA (PDB: 1HXD) with biotin (yellow) bound to the active site. Residue 118 is highlighted in red with the biotin binding site boxed in black. The panels to the right show the biotin binding site of BirA and the hydrogen bonds (black dotted lines) associated with Arg118 and Gly118. Modeling performed in PyMol. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Evaluation of auto-biotinylation by mutants of BirA at position 118. (A) Purification and biotinylation state (fl-neutra detection) of wild-type and mutant BirA proteins expressed as MBP fusions in E. coli. Each mutant protein contains a single amino acid substitution at position 118, as indicated. Equal amounts (1 μg) of each MBP-BirA mutant were analyzed by Coomassie blue staining and by blotting. (B) Scheme for Factor Xa mediated cleavage and analysis of auto-biotinylation. (C) Biotin detection within the MBP and BirA catalytic fragments of each BirA mutant (2.5 μg) before and after cleavage with Factor Xa. Blots were also probed with anti-MBP antibody. Bands corresponding to MBP-BirA, MBP, and BirA are indicated to the right of each panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Bead-based analysis of proximal biotinylation by BirA mutants. (A) Scheme for proximal (self and trans) biotinylation by BirA immobilized on an amylose bead surface. (B) Biotinylation reactions were performed with MBP-BirA mutants (5 μg) and free MPB (10 μg) on amylose beads in the presence of biotin (50 μM). Samples were analyzed by fl-neutra. Normalized biotinylation of self and trans labeling was quantified and plotted in (C).

Fig. 4.

Utilization of Biotin Acceptor Peptide (BAPs) for analyzing BirA mutants. (A) BAP sequences showing the position of the acceptor lysine and amino acid substitutions (bold underline). (B) In vitro biotinylation of BAP substrates with purified BirA enzymes. Reactions contained MBP-BirA (1 μg) with the indicated amount of each BAP and biotin (50 μM). (C) Quantification of relative biotinylation of GST-BAP (K- > A) in (B) indicates proximity labeling of a site outside of the BAP acceptor lysine. (D) TLC analysis of basal and BAP-induced bioAMP and AMP generation by BirA proteins. Assays were performed in triplicate and analyzed by autoradiography, one set of which is shown in this panel. (E) Quantification of AMP generation by BirA mutants. The % AMP is the percentage of signal in each lane corresponding to [α-³²P]-labeled AMP. (****p < 0.0001).

Fig. 5.

Evaluation of BirA biotin affinity and turn-over rate. TLC analysis of [α-³²P]-labeled bioAMP and AMP generation as a function of biotin concentration by (A) BirA, (B) BirA*, and (C) BirA(R118K). Signal for each [α-³²P]-labeled species was quantified using ImageJ and the data plotted as the ratio of AMP/bioAMP, a measure of the promiscuous release and hydrolysis of bioAMP to AMP in (G). (B, D, F) Biotinylation of BAP measured as a function of biotin concentration using fl-neutra. (H) BAP biotinylation is normalized to GST signal and plotted as a function of biotin concentration. Apparent K_M values calculated in GraphPad Prism are shown.

Fig. 6.

Cell-based experiments with BirA-Lamin A fusions. (A) Confocal microscopy showing the localization (red) and biotinylation (green) of BirA*-Lamin A and BirA(R118K)-Lamin A. HEK293T cells grown in the absence and presence of exogenous biotin (50 μM). Fluorescence intensities were measured across nuclei (white lines) and quantified (middle panels) (Scale bar 5 μm) (B) Whole cell lysate from HEK293T cells transfected with HA-Lamin A or BirA-Lamin A fusions with and without biotin supplementation (50 μM). Immunoblot of fl-neutra detection (arrow denotes BirA-Lamin A band) (C) Mass spectrometry results obtained from streptavidin pulldown of HEK293T lysate from cells expressing Lamin A-BirA(R118K) and Lamin A-BirA* in the absence of additional biotin supplementation. Shown are selected peptides derived from proteins known to be proximal to Lamin A in cells. A complete list of peptides from this analysis is provided (Table S3). (D) Localization of endogenous PCNA to the nuclear lamina by confocal fluorescence microscopy (Scale bar 5 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

Evaluation of BirA(R118K) activity after auto-modification. (A) Triton resistant fraction from HEK293T cells transfected, in the absence of biotin supplementation, with HA-Lamin A or BirA-Lamin A fusions were harvested and subsequently incubated with biotin for the indicated times. Immunoblot of fl-neutra detection as well (arrow denotes BirA-Lamin A band; asterisk denotes GST-BAP). Signal from fl-neutra on GST-BAP was normalized to GST for each lane and plotted in (B). (C) In vitro biotinylation of GST-BAP substrate (1 μg) with BirA(R118K) (0.2 μg) with and without a pre-incubation with 50 μM biotin for 2 h at 37 °C. Signal from fl-neutra on GST-BAP was normalized to GST for each lane and plotted in (D). (E) TLC based assay to evaluate production of [α-³²P]-labeled bioAMP and AMP by BirA, BirA* and BirA(R118K) after 90 min of in vitro biotinylation in the presence 50 μM biotin and 50 μM ATP (82.5 nM [α-³²P]-ATP) at 37 °C. (F) Quantification of the mols of product generated per mol of BirA enzyme based on a standard curve quantifying [α-³²P]-ATP signal per mol quantified by PhosphorImager (Fig. S2).