“Underappreciated” Flanking Regions Help Proteins Partner

Protein pairing is a highly specific process fundamental to cell function and regulation, but how proteins find their binding partners amid the billions of molecules within a cell is poorly understood. Since the wrong pairing—or the right pairing at the wrong place or time—can lead to disease, better  understanding of how the short amino acid segments that mediate protein-protein interactions could lead to new therapeutics.

Toward this end, biologists at MIT have published a pair of studies describing a new screening tool to probe the mechanisms by with these segments, called short linear motifs (SLiMs), recognize the correct binding partners and even distinguish between those with similar structures. They then created their own synthetic molecule capable of binding tightly to a protein implicated in cancer metastasis. The findings were reported in two recent studies published in eLife.

 “As we gain an understanding of the tricks that a protein uses to select its partners, we can apply these in protein design to make our own binders to modulate protein function for research or therapeutic purposes,” says Amy Keating, professor of biology and biological engineering and senior author on both studies.

Dubbed MassTitr, the MIT method is able to survey for SLiMs over a wider range of binding affinities than previous screening tools. The team also theorized that the amino acids on either side of SLiM’s core 4-6 amino acid sequence might play an “underappreciated" role in protein binding. To test this theory, they used MassTitr to see which “extended” SLiMs would associate with ENAH, a protein that helps cells move and can be hijacked by cancer cells to spread to other tissues.

By combining a computer program called dTERMen and X-ray crystallography, the team found that amino acids flanking the core SLiM in PCARE, a protein with high binding affinity for ENAH, caused ENAH to change shape slightly and allow binding. This explains why PCARE binds to ENAH instead of near-identical proteins VASP and EVL.

 “This work lays the foundation for designing synthetic molecules with the potential to disrupt protein-protein interactions that cause disease—or to help scientists learn more about ENAH and other SLiM-binding proteins,” says lead author Theresa Hwang.