Measuring Protein-Protein Interactions with Luminescent Proximity Assays

Identifying Inhibitors by High Throughput Screening

Luminescent proximity assays are commonly applied to high-throughput screening of drug libraries for inhibitors of known binding partners. For this application, the proteins are recombinantly expressed and bound to the donor and acceptor beads via antibodies or fusion tags (GST, 6xHis etc.) (Figure 1). When the proteins interact, the beads are brought together and emit light. A drug library can then be screened, with inhibitors of this interaction resulting in a reduction of signal. One example could include a histone modification reader such as a bromodomain bound to an acetylated peptide substrate; an inhibitor disrupts this interaction and results in a loss of luminescence. All of the specific applications described below can be adapted to screen for drug inhibitors of binding partners.

Figure 1: High-throughput screening for inhibitors of biomolecular interactions. In the general set up for this assay, streptavidin-coated donor beads are bound to a biotinylated protein, while Ni²⁺ chelate-coated acceptor beads are bound to a His-tagged protein (note that other methods of bead binding can be used). A library of small molecules is added, with inhibitors of this interaction resulting in reduced binding and a loss of signal.

Measuring Kinase Activity

There are a number of ways by which luminescent proximity assays can be applied to measuring enzyme activity. Though there are some generalized approaches to this, the exact format will depend on the specific enzyme being investigated and how it affects its substrate.

To measure kinase activity, there are two generalized approaches that may be taken (Figure 2):

1. Use two specific antibodies against the non-phosphorylated and phosphorylated forms of a protein as a proxy of kinase activity. The target can be either the full endogenous protein substrate, or a peptide containing a motif that becomes phosphorylated. The more enzyme activity, the more phosphorylated protein, the greater the signal emitted.
2. Bind a kinase substrate to donor beads, and the kinase itself to acceptor beads. Engagement of the substrate by the kinase will bring the beads together, producing signal. Like the previous example, the substrate attached to the donor beads can either be the full protein substrate or a simplified peptide domain.

Figure 2: Measuring kinase activity by luminescent proximity assay. A, Streptavidin-coated donor beads are bound to biotinylated anti-ERK1/2 antibodies, while Protein A-coated acceptor beads are bound to anti-phospho-ERK1/2 (pERK1/2) antibodies. B, A biotinylated polypeptide that contains a tyrosine to be phosphorylated is bound by streptavidin donor beads. Following the addition of the kinase and ATP, acceptor beads are brought into close proximity by anti-phospho-tyrosine antibodies. C, Streptavidin-coated donor beads are bound to biotinylated anti-substrate antibodies, while anti-kinase antibodies are conjugated directly to acceptor beads. D, Streptavidin donor beads bound to a biotinylated peptide substrate are brought close to acceptor beads via direct kinase activity.

The specific choice of format will depend on the experimental aims and set up, for example whether the experiments are being performed on cell lysates or cell-free systems. In the scenarios depicted in Figure 2, A and C use antibodies to detect endogenous interactions and so could be performed in cell lysates. In contrast, use of recombinant or purified proteins or peptides requires performing the experiment in vitro.

Phosphatase activity can also be measured with similar approaches, but starting out with phosphorylated substrates and looking for a reduction in signal upon addition of the phosphatase.

Measuring Protease Activity

Protease activity can be measured with luminescent proximity assay beads by looking for a reduction in signal after protein cleavage (Figure 3). A single protein substrate bridges the donor and acceptor beads by any number of means (specific antibodies to distinct epitopes, biotinylation, epitope tags etc.) to generate a light signal. Upon cleavage of the protein by the protease, the beads will separate and a loss of signal can be observed.

Figure 3: Measuring protease activity by luminescent proximity assay.

Luminescent proximity assays have a distinct advantage over comparable proximity-based approaches such as FRET in the analysis of protease activity, because energy transfer occurs across 200 nm compared to the ~9 nm in FRET. This means that the protein substrate can be much larger (e.g. a full-length endogenous protein) rather than a small peptide substrate.

Measuring Helicase Activity

To measure helicase activity, such as that of a DNA helicase, a double-stranded nucleic acid substrate is created where each strand carries a different molecular tag. For example, one strand might be biotinylated to bind streptavidin-coated donor beads, while the complementary strand is tagged with a 6xHis-tag or another epitope to be recognized by an acceptor bead–coupled antibody (Figure 4).

When the DNA is intact (double stranded), the two beads are close and emit light at 615 nm. After the addition of helicase and ATP, the strands unwind and the beads separate, resulting in a drop in signal. Like the other assays described on this page, this set up can then be easily applied to high-throughput screening to search for inhibitors of this interaction.

Figure 4: Helicase activity measured by luminescent proximity assay.

Measuring Ubiquitin Ligase Activity

Ubiquitin ligases attach ubiquitin as a posttranslational modification to proteins in order to target them for proteasomal degradation. Measuring the activity of ubiquitin ligases can be achieved with biotinylated ubiquitin. Biotinylated ubiquitin is bound by streptavidin donor beads, while other proteins are bound to acceptor beads via a tag, such as GST (Figure 5). When a protein is ubiquitinated, the beads will be brought into proximity to produce a signal. This approach can be used to find modulators of this activity, or even to identify novel substrates for the ligase.²

Figure 5: Ubiquitin ligase activity measured by luminescent proximity assay.

Characterization of PROTAC

A proteolysis targeting chimera (PROTAC) is a molecule used to target proteins of interest for ubiquitination, resulting in their degradation by the proteasome.³ The PROTAC molecule itself consists of two binding domains: one that binds to the protein of interest, and another that engages with an E3 ubiquitin ligase. When brought together, the ligase polyubiquitinates the target protein.

Luminescent proximity assays are ideal for characterizing PROTACs both in vitro and in cells. For instance, PROTAC molecules can be quickly assessed for their ability to bring the protein of interest and the E3 ligase into close proximity (Figure 6). A tagged E3 ligase is bound to donor beads, while the protein of interest is fused to a different tag and bound to acceptor beads. The presence of a specific PROTAC brings the beads into close proximity, resulting in emission at 615 nm.

Figure 6: Measuring PROTAC complex formation with recombinant proteins. E3 ligase is GST-tagged and bound by anti-GST donor beads, while a protein of interest is His-tagged and bound by Ni²⁺ chelate-coated acceptor beads. Addition of the PROTAC molecule binds to both proteins and brings the beads into proximity.

Other aspects of PROTAC can also be investigated using luminescent proximity assays, including:

• E2 recruitment: E2 needs to be recruited to the complex for polyubiquitination of the protein of interest to occur. To measure this, biotinylated ubiquitin bridging streptavidin donor beads and E2 is brought to the ternary complex, with the tagged protein of interest bound to acceptor beads.
• Polyubiquitination: Tandem ubiquitin binding entities (TUBES) are engineered proteins that bind to polyubiquitin chains. The interaction between biotinylated TUBES (on streptavidin donor beads) and the ubiquitinated protein of interest (on acceptor beads) indicates the extent of protein ubiquitination.
• Specific protein degradation: Donor and acceptor beads can be brought together by a single protein, as outlined in ELISA-like Luminescent Proximity Assays for Single Analytes, by using two antibodies against distinct epitopes, like in a sandwich ELISA. If the protein of interest is being degraded, the beads will cease to be in close proximity and a reduction of signal will be seen. This approach can be performed in cells or in cell-free systems.

Identifying Novel Binding Partners

As well as finding inhibitors of known interactions, luminescent proximity assays are also useful for identifying novel binding partners of a specific target. The target is biotinylated and bound to streptavidin donor beads, while a library of potential binding partners can be expressed with a tag (e.g. FLAG tag) and bound by antibody-coated (e.g. anti-FLAG antibodies) acceptor beads. Creating a library of full-length proteins would be costly, time-consuming and labor-intensive, but a library of shorter peptide fragments representing important binding motifs can be used instead.

Phage Display

Phage display screening technology can be combined with luminescent proximity assays for a highly sensitive approach to identifying strong binding partners (Figure 7). The bead assay is integrated after biopanning, once a pool of potential binding partners has been identified, replacing other validation steps such as ELISA. Combing the approaches in this way has several advantages, including:

1. Validation of interactions and reducing false positives, because biopanning can produce weak or non-specific interactions
2. Quantitative measurement of the strength of binding to identify high affinity binders
3. Screening binders in solution, which may be more physiologically relevant than when one partner is immobilized to a plate
4. Scalability and efficiency, because bead-based luminescent proximity assays are suited to high-throughput screening in case of a large number of positive phages.

As before, the target is biotinylated and bound to streptavidin-coated donor beads, while the acceptor beads are conjugated to anti-M13 antibodies, which bind to phage coat proteins. When a phage expresses a peptide that can interact with the target, the beads are brought together to produce a signal. Similar to normal phage display screening, this process can be repeated multiple times to identify the highest affinity binding partners.

Figure 7: Combining phage display with luminescent proximity assays to identify novel binding partners. A phage display library is created containing peptides, which are then tested against the target immobilized to a solid surface. Unbound phages are washed away, allowing bound phages to be subsequently eluted. After repeating this biopanning process several times, potential binding peptides are assessed by luminescent proximity assay, binding the phages with anti-M13 antibodies.