Proximity Tagging of Protein-Protein Interactions

There are four research areas in the Department of Pharmaceutical Chemistry. Proximity tagging of protein-protein interactions is a research challenge within protein and cellular engineering.

The challenge

Thousands of different protein molecules in our cells are constantly interacting, altering each other’s chemical forms and functions in ways that underlie both healthy homeostasis and the dysregulation of disease. One way to keep track of those dynamic interactions (collectively known as the interactome), which may consist of only weak binding and be extremely short-lived, is to engineer enzymes to transfer labels so transient complexes and networks can be detected and quantified.

Examples of our research and methods include

Identifying proteins that inhibit apoptosis

Ubiquitination is a common post-translational modification of more than 5,000 proteins involved in a variety of cellular processes. The final step, catalyzed by E3 ubiquitin ligases, tags proteins with ubiquitin, which labels them for rapid destruction by protein complexes called proteasomes.

Apoptosis is the programmed self-destruction of aberrant cells that is crucial to normal health. It fails to occur in cancer and can occur too much, as in neurodegenerative diseases.

A subset of E3 ligases act as inhibitors of apoptosis proteins (IAPs), targeting certain caspases, cysteine proteases that carry out the destruction of cells, as well as caspases’ numerous protein allies, which act as systemic counterbalances, preventing IAPs from binding to caspases.

To better understand the protein networks involved in cellular apoptosis, researchers here use engineered enzymes to proximity tag proteins in order to identify IAP substrates.

Our applications of such protein engineering include

Labeling weak, transient protein interactions

Re-engineering an IAP ubiquitin ligase by genetically fusing its substrate-binding domain with another enzyme that instead covalently attaches a rare ubiquitin homolog, NEDD8. This allowed more than 50 IAP natural substrates to be identified and quantified by mass spectrometry.

wild type RING E3 ligase with NEDDylator
  • A: A natural (wild-type) E3 ubiquitin ligase (green) joins with an E2 ubiquitin-conjugating enzyme (cyan/bright blue), then recognizes and binds substrate (S) with its substrate binding domain (SBD) and transfers a ubiquitin protein (or chains of ubiquitin) to a substrate lysine residue, labeling it for degradation or otherwise altering its activity.
  • B: In the engineered version (NEDDylator), an enzyme conjugating NEDD8 (orange) is fused to the ligase substrate binding domain, labeling the substrate for identification and quantification via mass spectrometry.

More generally, the fused enzyme, dubbed the NEDDylator, demonstrated a robust method for labeling weak and transient protein-protein binding partners such as those in E3 substrate interactions in ubiquitination.