Exploiting ligand-protein conjugates to monitor ligand-receptor interactions

Abstract

We introduce three assays for analyzing ligand-receptor interactions based on the specific conjugation of ligands to SNAP-tag fusion proteins. Conjugation of ligands to different SNAP-tag fusions permits the validation of suspected interactions in cell extracts and fixed cells as well as the establishment of high-throughput assays. The different assays allow the analysis of strong and weak interactions. Conversion of ligands into SNAP-tag substrates thus provides access to a powerful toolbox for the analysis of their interactions with proteins.

Figure 1. SNAP-based toolbox for detection and analysis of ligand-receptor interactions.

(A) Covalent labeling of SNAP-tag with a ligand using a BG derivative. (B) Schematic representation of the different SNAP-based methods. The highest Kd values detected in this study using the pairs of MTX-eDHFR (WT and mutants) are presented for each method.

Figure 2. SNAP-based TR-FRET titration assay.

(A) Scheme of the titration binding assays. The titration assay measures the affinity of the tracer for the receptor. (B) Titration assay using MTX-SNAP-EGFP (filled rectangle) or SNAP-EGFP (empty rectangle) as tracer and SNAP-eDHFR as receptor in the absence and presence of 100 µM NADPH. SNAP-eDHFR is 50% labeled with BG-Terbium cryptate (Tb). Representative data using receptor concentration of 1 nM (in the absence of NADPH) and 0.1 nM (in the presence of NADPH) are shown. The specific receptor concentration was chosen so that it was below the Kd of the analyzed interaction. Kd values and the standard error of the mean are shown in the graph. * indicates that the Kd values were calculated with Fmax of the higher affinity samples (see Materials and Methods).

Figure 3. SNAP-based TR-FRET competition assay.

(A) Scheme of the competition binding assay. The competition assay measures the affinity of the ligand for the receptor. (B) Chemical structures of DHFR inhibitors. MTX was linked to BG via the carboxyl group highlighted in red. (C) SNAP-based TR-FRET competition assays using indicated concentrations of SNAP-eDHFR WT, L54I and L54G. Concentrations of MTX-SNAP-EGFP are 1 nM, 2 nM, and 30 nM for SNAP-eDHFR WT, L54I and L54G, respectively. Rectangle filled with black indicates dilution series of DMSO. Maximum DMSO concentration is 0.5% at 100 µM compounds. (D) DHFR enzymatic activity inhibition assay using SNAP-eDHFR WT.

Figure 4. SNAP-based TR-FRET competition assay for ORP7-erlotinib interaction.

The assay was performed at indicated concentrations of SNAP-ORP7 (receptor) and erlotinib-SNAP-EGFP (tracer) with erlotinib as a competitor. IC50 values and the standard error of the mean are shown.

Figure 5. SNAP-based pulldown assay for detection of ligand-receptor interactions.

(A) Scheme of the pulldown assay. The BG-ligand is immobilized on glutathione sepharose beads via GST-SNAP. Bound receptor proteins are concentrated on the beads after incubation with the extract and washing. (B) SNAP-eDHFR WT labeled with BG-647 (1 µM) was subjected to pulldown assay in the absence or presence of 100 µM NADPH using MTX immobilized on beads (MTX-BG +) or mock beads (MTX-BG -). Bound proteins were eluted with glutathione, submitted to SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of bound proteins normalized with the input signal in each gel (Mean±SD, n = 3). # represents P = 0.03 in paired t-test.

Figure 6. S-CROSS assay for the detection of ligand-receptor interactions.

(A) Scheme of the S-CROSS assay. SNAP-receptor and ligand-SNAP-CLIP are incubated to form a complex. BG-fluorophore-BC preferentially crosslinks proteins that are in close spatial proximity. (B) SNAP-eDHFR WT (1 µM) was mixed with MTX-SNAP-CLIP or unlabeled SNAP-CLIP (1 µM) in the presence or absence of free MTX (50 µM). The experiments were performed in the absence or presence of 100 µM NADPH. Then, the mixture was treated with 2.5 µM BG-647-BC. Labeled proteins were resolved by SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of the hetero-crosslinking products in the presence or absence of free MTX (Mean±SD, n = 3–5). # represents P = 0.001 in paired t-test. (D) S-CROSS assay in cell extract. SNAP-ORP7 was expressed in HEK293 cells. After preparation of extract, SNAP-ORP7 was subjected to S-CROSS assay using erlotinib-SNAP-CLIP (2 µM) in the absence or presence of free erlotinib (10 µM).

Figure 7. Cell imaging assay for detection of ligand-receptor interactions.

(A) Scheme of the cell imaging assay. The receptor protein is transiently expressed in mammalian cells. Cells are fixed with 4% paraformaldehyde, and then permeabilized with detergent. Ligand-SNAP-EGFP is incubated with the cells. Immediately after washing, samples are analyzed with a fluorescence microscope. (B) Fluorescence micrographs of U2OS cells transiently expressing V5-ORP7 probed with FITC-conjugated anti-V5 antibody (left) and erlotinib-SNAP-EGFP (right) by cell imaging assay (FITC filter in green). Nuclear staining with Hoechst 33342 is shown in blue (UV filter).