Checking how proteins pair up inside cells

Short linear figures (blue) use surrounding regions outside the essential amino acid sequences to identify proteins such as ENAH (in grey). Credit: Theresa Hwang

Despite its small size, a single cell contains billions of molecules that wrestle around and bind to each other, performing vital functions. The human genome encodes approximately 20,000 proteins, most of which interact with partner proteins to mediate over 400,000 distinct interactions. These partners don’t stick to each other randomly; They only stick to very specific companions that they must get to know within the crowded dungeon. If they create the wrong pairs—or even the right pairing in the wrong place or at the wrong time—cancer or other diseases can occur. Scientists are working hard to investigate these protein-protein relationships, in order to understand how they work, and potentially make drugs that disrupt or mimic them in treating diseases.

The average human protein is made up of about 400 building blocks called amino acids, which are joined together and folded into a complex three-dimensional structure. Within this long chain of building blocks, some proteins contain stretches of 4-6 amino acids called short linear motifs (SLiMs), which mediate protein-protein interactions. Despite their simplicity and small size, SLiMs and their binding partners facilitate key cellular processes. However, it has historically been difficult to conduct experiments to verify how SLiMs recognize their specific binding partners.

To address this problem, a group led by Theresa Hwang Ph.D. ’21 designed a screening method to understand how SLiMs selectively bind to specific proteins, and even distinguish those with similar structures. Using the detailed information they obtained from studying these interactions, the researchers created their own synthetic molecule capable of binding very tightly to a protein called ENAH, which is implicated in cancer metastasis. The team shared their findings in a pair of eLife One was published on January 25, 2022, and the other on December 2, 2021.

“The ability to test hundreds of thousands of potential SLiMs for binding provides a powerful tool for exploring why proteins prefer certain SLiM partners over others,” says Amy Keating, professor of biology and biological engineering and lead author on both studies. “When we understand the tricks a protein uses to choose its partners, we can apply them in protein design to make our own bonds to modulate protein function for research or therapeutic purposes.”

Most screens on SLiMs simply choose short and tight bonds, while ignoring SLiMs that do not hold their partner proteins as strongly. To scan SLiMs with a wide range of ligands, Keating, Hwang and their colleagues developed their own screen called MassTitr.

The researchers also suspected that the amino acids on either side of the 4-6 essential amino acid sequence of SLiM might play a truly underestimated role in the binding. To test their theory, they used MassTitr to examine the human protein in longer fragments made up of 36 amino acids, in order to see which “stretched” SLiMs would bind to the protein ENAH.

ENAH, sometimes referred to as Mena, helps cells move. This ability to migrate is critical for healthy cells, but cancer cells can metastasize. Scientists have found that reducing the amount of ENAH reduces the ability of cancer cells to invade other tissues — suggesting that formulating drugs to disrupt this protein and its interactions could treat cancer.

Thanks to MassTitr, the team identified 33 SLiM-containing proteins associated with ENAH – 19 of which are potentially new binding partners. They also discovered three distinct amino acid patterns flanking the core SLiM sequences that helped the SLiMs bind more tightly to ENAH. Among these extended SLiMs, there is one in a protein called PCARE bound to ENAH with the highest affinity known of any SLiM to date.

Next, the researchers combined a computer program called dTERMen with X-ray crystallography in order to understand how and why PCARE binds to ENAH via two nearly identical sister proteins to ENAH (VASP and EVL). Huang and colleagues saw that the amino acids surrounding the SliM essential for PCARE caused ENAH to change slightly when the two contacted, allowing the binding sites to stick together. By contrast, VASP and EVL could not undergo this structural change, so neither PCARE SliM adhered tightly.

Inspired by this unique interaction, Hwang engineered her own ENAH-binding protein with unprecedented affinity and specificity. “It was exciting that we were able to devise such a specific link,” she says. “This work lays the foundation for the design of synthetic molecules that have the ability to disrupt protein-protein interactions that cause disease—or to help scientists learn more about ENAH and other SLiM-related proteins.”

Understanding how proteins find their binding partners is an issue of fundamental importance to cell function and regulation, says Elva Ivarsson, a professor of biochemistry at Uppsala University who was not involved in the study. the two eLife Studies demonstrate that extended SLiMs play an underappreciated role in determining the affinity and specificity of these binding interactions.

“The studies highlight the idea that context is important, and provide a screening strategy for a variety of context-dependent binding interactions,” she says. “Hwang and co-authors have created valuable tools to dissect the cellular function of proteins and their binding partners. Their approach could even inspire specific ENAH inhibitors for therapeutic purposes.”

Hwang’s biggest benefit from the project is that things aren’t always what they seem: Even short, simple protein segments can play complex roles in a cell. In her words: “We should really appreciate the SLiMs more.”

Breakdown of 3D Protein Structures: Linked as a Loop

more information:
The native proline-rich polymorphisms exploit the sequence context to target the Ena/VASP protein ENAH, eLife, DOI: 10.7554/eLife.70680,

The network of distributed residues allows specificity of conformational binding in a conserved family of actin remodelers, eLife, DOI: 10.7554/eLife.70601,

Journal information:

Submitted by MIT, Department of Biology

the quote: Investigating how proteins pair up inside cells (2022, January 25) Retrieved on January 26, 2022 from

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